Processes for producing industrial products from plant lipids

ABSTRACT

The present invention relates to methods of producing industrial products from plant lipids, particularly from vegetative parts of plants. In particular, the present invention provides oil products such as biodiesel and synthetic diesel and processes for producing these, as well as plants having an increased level of one or more non-polar lipids such as triacylglycerols and an increased total non-polar lipid content. In one particular embodiment, the present invention relates to combinations of modifications in two or more of lipid handling enzymes, oil body proteins, decreased lipid catabolic enzymes and/or transcription factors regulating lipid biosynthesis to increase the level of one or more non-polar lipids and/or the total non-polar lipid content and/or mono-unsaturated fatty acid content in plants or any part thereof. In an embodiment, the present invention relates to a process for extracting lipids. In another embodiment, the lipid is converted to one or more hydrocarbon products in harvested plant vegetative parts to produce alkyl esters of the fatty acids which are suitable for use as a renewable biodiesel fuel.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/793,663, filed Jul. 7, 2015, now allowed, and claims priority ofAustralian patent Applications Nos. 2014902617, filed Jul. 7, 2014;2015900084, filed Jan. 13, 2015; and 2015900284, filed Jan. 30, 2015,the contents of each of which are hereby incorporated by reference intheir entirety.

REFERENCE TO A SEQUENCE LISTING

This application incorporates-by-reference nucleotide and/or amino acidsequences which are present in the file named“191107_87776-A_Sequence_Listing_CAS.txt”, which is 668 kilobytes insize, and which was created Nov. 7, 2019 in the IBM-PC machine format,having an operating system compatibility with MS-Windows, which iscontained in the text file filed Nov. 7, 2019 as part of thisapplication.

FIELD OF THE INVENTION

The present invention relates to methods of producing industrialproducts from plant lipids, particularly from vegetative parts ofplants. In particular, the present invention provides oil products suchas biodiesel and synthetic diesel and processes for producing these, aswell as plants having an increased level of one or more non-polar lipidssuch as triacylglycerols and an increased total non-polar lipid content.In one particular embodiment, the present invention relates tocombinations of modifications in two or more of lipid handling enzymes,oil body proteins, decreased lipid catabolic enzymes and/ortranscription factors regulating lipid biosynthesis to increase thelevel of one or more non-polar lipids and/or the total non-polar lipidcontent and/or mono-unsaturated fatty acid content in plants or any partthereof. In an embodiment, the present invention relates to a processfor extracting lipids. In another embodiment, the lipid is converted toone or more hydrocarbon products in harvested plant vegetative parts toproduce alkyl esters of the fatty acids which are suitable for use as arenewable biodiesel fuel.

BACKGROUND OF THE INVENTION

The majority of the world's energy, particularly for transportation, issupplied by petroleum derived fuels, which have a finite supply.Alternative sources which are renewable are needed, such as frombiologically produced oils.

Triacylglycerol Biosynthesis

Triacylglycerols (TAG) constitute the major form of lipids in seeds andconsist of three acyl chains esterified to a glycerol backbone. Thefatty acids are synthesized in the plastid as acyl-acyl carrier protein(ACP) intermediates where they may undergo a first desaturationcatalyzed. This reaction is catalyzed by the stearoyl-ACP desaturase andyields oleic acid (C18:1^(Δ9)). Subsequently, the acyl chains aretransported to the cytosol and endoplasmic reticulum (ER) asacyl-Coenzyme (CoA) thioesters. Prior to entering the major TAGbiosynthesis pathway, also known as the Kennedy or glycerol-3-phosphate(G3P) pathway, the acyl chains are typically integrated intophospholipids of the ER membrane where they can undergo furtherdesaturation. Two key enzymes in the production of polyunsaturated fattyacids are the membrane-bound FAD2 and FAD3 desaturases which producelinoleic (C18:2^(Δ9,12)) and α-linolenic acid (C18:3^(Δ9,12,15))respectively.

TAG biosynthesis via the Kennedy pathway consists of a series ofsubsequent acylations, each using acyl-CoA esters as the acyl-donor. Thefirst acylation step typically occurs at the sn1-position of the G3Pbackbone and is catalyzed by the glycerol-3-phosphate acyltransferase(sn1-GPAT). The product, sn1-lysophosphatidic acid (sn1-LPA) serves as asubstrate for the lysophosphatidic acid acyltransferase (LPAAT) whichcouples a second acyl chain at the sn2-position to form phosphatidicacid. PA is further dephosphorylated to diacylglycerol (DAG) by thephosphatidic acid phosphatase (PAP) thereby providing the substrate forthe final acylation step. Finally, a third acyl chain is esterified tothe sn3-position of DAG in a reaction catalyzed by the diacylglycerolacyltransferase (DGAT) to form TAG which accumulates in oil bodies. Asecond enzymatic reaction, phosphatidyl glycerol acyltransferase (PDAT),also results in the conversion of DAG to TAG. This reaction is unrelatedto DGAT and uses phospholipids as the acyl-donors.

To maximise yields for the commercial production of lipids, there is aneed for further means to increase the levels of lipids, particularlynon-polar lipids such as DAGs and TAGs, in transgenic organisms or partsthereof such as plants, seeds, leaves, algae and fungi. Attempts atincreasing neutral lipid yields in plants have mainly focused onindividual critical enzymatic steps involved in fatty acid biosynthesisor TAG assembly. These strategies, however, have resulted in modestincreases in seed or leaf oil content. Recent metabolic engineering workin the oleaginous yeast Yarrowia lipolytica has demonstrated that acombined approach of increasing glycerol-3-phosphate production andpreventing TAG breakdown via β-oxidation resulted in cumulativeincreases in the total lipid content (Dulermo et al., 2011).

Plant lipids such as seedoil triaclyglycerols (TAGs) have many uses, forexample, culinary uses (shortening, texture, flavor), industrial uses(in soaps, candles, perfumes, cosmetics, suitable as drying agents,insulators, lubricants) and provide nutritional value. There is alsogrowing interest in using plant lipids for the production of biofuel.

To maximise yields for the commercial biological production of lipids,there is a need for further means to increase the levels of lipids,particularly non-polar lipids such as DAGs and TAGs, in transgenicorganisms or parts thereof such as plants, seeds, leaves, algae andfungi.

SUMMARY OF THE INVENTION

The present inventors have identified a process for producing an oilproduct from vegetative plant parts.

In a first aspect, the present invention provides a process forproducing an oil product, the process comprising the steps of

(i) treating, in a reactor, a composition comprising

-   -   (a) vegetative plant parts whose dry weight is at least 2 g and        which have a total non-polar lipid content of at least 5% by        weight on a dry weight basis,    -   (b) a solvent which comprises water, an alcohol, or both, and    -   (c) optionally a catalyst,        wherein the treatment comprises heating the composition at a        temperature between about 50° C. and about 450° C. and at a        pressure between 5 and 350 bar for between 1 and 120 minutes in        an oxidative, reductive or inert environment,

(ii) recovering oil product from the reactor at a yield of at least 35%by weight relative to the dry weight of the vegetative plant parts,thereby producing the oil product.

In an embodiment, the vegetative plant parts have a dry weight of atleast 1 kg.

In an embodiment, the vegetative plant parts have a total non-polarlipid content of at least 10%, at least 15%, at least 20%, about 25%,about 30%, about 35%, between 10% and 75%, between 20% and 75% orpreferably between 30% and 75% on a dry weight basis.

In an embodiment, the composition has a solids concentration between 5%and 90%, preferably between 15% and 50% (dry weight/weight).

Any suitable catalyst can be used. In an embodiment, the catalyst is analkali, an acid or a precious metal catalyst. For instance, in anembodiment the catalyst comprises NaOH or KOH or both, preferably at aconcentration of 0.1M to 2M.

In an embodiment, the treatment time is between 1 and 60 minutes,preferably between 10 and 60 minutes, more preferably between 15 and 30minutes. In an embodiment where the pressure is less than 50 bar, thetime of reaction may be up to 24 hours or even up to 7 days. In apreferred embodiment, the temperature is between 275° C. and 360° C.,the pressure is between 100 and 200 bar, and the reaction occurs in10-60 mins.

In an embodiment, if the solvent is water the process produces a yieldof the oil product between a minimum of 36%, 37%, 38%, 39% or 40% and amaximum of 55% or preferably 60% by weight relative to the dry weight ofthe vegetative plant parts. In this embodiment, the oil productcomprises at least 2-fold, preferably at least 3-fold more hydrocarboncompounds than fatty acyl esters. Preferably, the oil product comprises35%, more preferably 40% C13-C22 hydrocarbon compounds.

In another embodiment, if the solvent comprises an alcohol, preferablymethanol, the process produces a yield of the oil product between aminimum of 36%, 37%, 38%, 39% or 40% and a maximum of 65% or preferably70% by weight relative to the dry weight of the vegetative plant parts.In this embodiment, the oil product comprises at least 1.5-fold,preferably at least 2-fold, more fatty acyl esters than hydrocarboncompounds. Preferably, the oil product comprises 40%, more preferably50%, fatty acid methyl esters.

In a further embodiment, if the solvent comprises about 80% water, theoil product comprises about 30% of C13-C22 hydrocarbon compounds,preferably about 35%, more preferably about 40% C13-C22 hydrocarboncompounds.

In another embodiment, if the solvent comprises about 50% methanol, theoil product comprises about 50% fatty acid methyl esters (FAME).

In a further embodiment, the recovered oil product has a water contentof less than about 15% by weight, preferably less than 5% by weight.

In yet another embodiment, the yield of oil product is at least 2%greater by weight, preferably at least 4% greater by weight, relative toa corresponding process using corresponding vegetative plant parts whosenon-polar lipid content is less than 2% on a dry weight basis.

In an embodiment, the vegetative plant parts in step (i)(a) have beenphysically processed by one or more of drying, chopping, shredding,milling, rolling, pressing, crushing or grinding. In an alternativeembodiment, the vegetative plant parts have not been dried to a moisturecontent of less than 10% prior to preparation of the composition. Forexample, the vegetative plant parts have a moisture content of at least20% or at least 30%, or the vegetative plant parts retain at least 50%of the water content that they had at the time they were harvested.

In an embodiment, the process further comprises one or more of:

(i) hydrodeoxygenation of the recovered oil product,

(ii) treatment of the recovered oil product with hydrogen to reduce thelevels of ketones or sugars in the oil product,

(iii) production of syngas from the recovered oil product, and

(iv) fractionating the recovered oil product to produce one or more offuel oil, diesel oil, kerosene or gasoline. For example, thefractionating step is by fractional distillation.

In an embodiment, the vegetative plant parts comprise plant leaves,stems or both.

In an embodiment, the vegetative plant parts comprise a combination ofexogenous polynucleotides and/or genetic modifications as definedherein.

The present inventors have also demonstrated significant increases inthe lipid content of organisms, particularly in the vegetative parts andseed of plants, by manipulation of fatty acid biosynthesis, lipidassembly and lipid packaging pathways, and reduced lipid catabolism.Various combinations of genes and reduction of gene expression were usedto achieve substantial increases in oil content, which is of greatsignificance for production of biofuels and other industrial productsderived from oil.

In a second aspect, the present invention provides a recombinanteukaryotic cell comprising

a) first exogenous polynucleotide which encodes a transcription factorpolypeptide that increases the expression of one or more glycolyticand/or fatty acid biosynthetic genes in the cell,

b) a second exogenous polynucleotide which encodes a polypeptideinvolved in the biosynthesis of one or more non-polar lipids, and anyone or two or all three of

c) a first genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in the catabolismof triacylglycerols (TAG) in the cell when compared to a correspondingcell lacking the genetic modification,

d) a third exogenous polynucleotide which encodes a polypeptide whichincreases the export of fatty acids out of plastids of the cell whencompared to a corresponding cell lacking the fourth exogenouspolynucleotide, and

e) a fourth exogenous polynucleotide which encodes a secondtranscription factor polypeptide that increases the expression of one ormore glycolytic and/or fatty acid biosynthetic genes in the cell,

wherein each exogenous polynucleotide is operably linked to a promoterwhich is capable of directing expression of the polynucleotide in thecell.

In an embodiment, the cell comprises a), b) and c), and optionally d) ore).

In an embodiment, the cell comprises a), b) and d), and optionally c) ore).

In an embodiment, the cell comprises a), b) and e), and optionally c) ord).

In an embodiment, the cell further comprises one or more or all of

a) a fifth exogenous polynucleotide which encodes an oil body coating(OBC) polypeptide, preferably a lipid droplet associated protein (LDAP),

b) a second genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in importing fattyacids into plastids of the cell when compared to a corresponding celllacking the second genetic modification, and

c) a third genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in diacylglycerol(DAG) production in the plastid when compared to a corresponding celllacking the third genetic modification.

In an embodiment, the recombinant eukaryotic cell comprises

a) a first exogenous polynucleotide which encodes a transcription factorpolypeptide that increases the expression of one or more glycolyticand/or fatty acid biosynthetic genes in the cell,

b) a second exogenous polynucleotide which encodes a polypeptideinvolved in the biosynthesis of one or more non-polar lipids, and

c) a first genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in the catabolismof triacylglycerols (TAG) in the cell when compared to a correspondingcell lacking the genetic modification,

wherein each exogenous polynucleotide is operably linked to a promoterwhich is capable of directing expression of the polynucleotide in thecell, and optionally the cell further comprises one or more or all of

d) a third exogenous polynucleotide which encodes an oil body coating(OBC) polypeptide, preferably an LDAP,

e) a fourth exogenous polynucleotide which encodes a polypeptide whichincreases the export of fatty acids out of plastids of the cell whencompared to a corresponding cell lacking the fourth exogenouspolynucleotide,

f) a second genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in importing fattyacids into plastids of the cell when compared to a corresponding celllacking the second genetic modification, and

g) a third genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in diacylglycerol(DAG) production in the plastid when compared to a corresponding celllacking the third genetic modification.

In an embodiment, the cell is a plant cell from or in a vegetative partof a plant and one or more or all of the promoters are expressed at ahigher level in the vegetative part relative to seed of the plant.

In a preferred embodiment, the presence of the c), d) or e), togetherwith the first and second exogenous polynucleotides increases the totalnon-polar lipid content of the cell, preferably a cell in vegetativeplant part such as a leaf or stem, relative to a corresponding cellwhich comprises the first and second exogenous polynucleotides butlacking each of c), d) and e). More preferably, the increase issynergistic. Most preferably, at least the promoter that directsexpression of the first exogenous polynucleotide is a promoter otherthan a constitutive promoter.

In an embodiment, the polypeptide involved in the biosynthesis of one ormore non-polar lipids is a DGAT or a PDAT and the polypeptide involvedin the catabolism of TAG in the cell is an SDP1 lipase.

In an embodiment, the transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the cell is a WRI1 polypeptide and the polypeptide involved inthe biosynthesis of one or more non-polar lipids is a DGAT or a PDAT.

In an embodiment, the transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the cell is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1polypeptide or a LEC1-like polypeptide and the polypeptide involved inthe biosynthesis of one or more non-polar lipids is a DGAT.

In an embodiment, the transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the cell is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1polypeptide or a LEC1-like polypeptide and the polypeptide involved inthe catabolism of triacylglycerols (TAG) in the cell is an SDP1 lipase.

In an embodiment, the transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the cell is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1polypeptide or a LEC1-like polypeptide, the polypeptide involved in thebiosynthesis of one or more non-polar lipids is a DGAT or a PDAT and thepolypeptide involved in the catabolism of triacylglycerols (TAG) in thecell is an SDP1 lipase.

In an embodiment, when present, the two transcription factors are WRI1and LEC2, or WRI1 and LEC1.

In the above embodiments, it is preferred that the cell is in avegetative part of a plant which is growing in soil or which was grownin soil and the plant part was subsequently harvested, and wherein thecell comprises at least 8% TAG on a weight basis (% dry weight) such asfor example between 8% and 75% or between 8% and 30%. More preferably,the TAG content is at least 10%, such as for example between 10% and 75%or between 10% and 30%. Preferably, these TAG levels are present in thevegetative parts prior to or at flowering of the plant or prior to seedsetting stage of plant development. In these embodiments, it ispreferred that the ratio of the TAG content in the leaves to the TAGcontent in the stems of the plant is between 1:1 and 10:1, and/or theratio is increased relative to a corresponding cell comprising the firstand second exogenous polynucleotides and lacking the first geneticmodification.

In the above embodiments, the cell preferably comprises an exogenouspolynucleotide which encodes a DGAT and a genetic modification whichdown-regulates production of an endogenous SDP1 lipase. More preferably,the cell does not comprise an exogenous polynucleotide encoding a PDAT,and/or is a cell other than a Nicotiana benthamiana cell, and/or theWRI1 is a WRI1 other than Arabidopsis thaliana WRI1 (SEQ ID NOs:21 or22). Most preferably, at least one of the exogenous polynucleotides inthe cell is expressed from a promoter which is not a constitutivepromoter such as, for example, a promoter which is expressedpreferentially in green tissues or stems of the plant or that isup-regulated after commencement of flowering or during senescence.

In a third aspect, the present invention provides a recombinanteukaryotic cell comprising

a) a first exogenous polynucleotide which encodes a transcription factorpolypeptide that increases the expression of one or more glycolyticand/or fatty acid biosynthetic genes in the cell,

b) a second exogenous polynucleotide which encodes a polypeptideinvolved in the biosynthesis of one or more non-polar lipids, and

c) a third exogenous polynucleotide which encodes an oil body coating(OBC) polypeptide, preferably a lipid droplet associated polypeptide(LDAP),

wherein each exogenous polynucleotide is operably linked to a promoterwhich is capable of directing expression of the polynucleotide in thecell, and wherein the recombinant eukaryotic cell has an increased levelof one or more non-polar lipid(s), and/or an increased amount of the OBCpolypeptide, relative to a corresponding cell which comprises a thirdexogenous polynucleotide whose nucleotide sequence is the complement ofthe sequence provided as SEQ ID NO: 176.

In an embodiment, the cell of the above aspect further comprises one ormore or all of

d) a first genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in the catabolismof triacylglycerols (TAG) in the cell when compared to a correspondingcell lacking the first genetic modification,

e) a fourth exogenous polynucleotide which encodes a polypeptide whichincreases the export of fatty acids out of plastids of the cell whencompared to a corresponding cell lacking the fourth exogenouspolynucleotide,

f) a second genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in importing fattyacids into plastids of the cell when compared to a corresponding celllacking the second genetic modification, and

g) a third genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in diacylglycerol(DAG) production in the plastid when compared to a corresponding celllacking the third genetic modification.

In an embodiment, the polypeptide involved in the biosynthesis of one ormore non-polar lipids is a DGAT or a PDAT and the OBC polypeptide is anoleosin. Alternatively, the OBC polypeptide is an LDAP.

In an embodiment, the transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the cell is a WRI1 polypeptide and the polypeptide involved inthe biosynthesis of one or more non-polar lipids is a DGAT or a PDAT.

In an embodiment, the transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the cell is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1polypeptide or a LEC1-like polypeptide and the OBC polypeptide is anoleosin. Alternatively, the OBC polypeptide is an LDAP.

In an embodiment, the transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the cell is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1polypeptide or a LEC1-like polypeptide, the polypeptide involved in thebiosynthesis of one or more non-polar lipids is a DGAT or a PDAT and theOBC polypeptide is an oleosin. Alternatively, the OBC polypeptide is anLDAP.

In an embodiment, the cell comprises two exogenous polynucleotidesencoding two different transcription factor polypeptides that increasethe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the cell, such as WRI1 and LEC2, or WRI1 and LEC1.

In a preferred embodiment, the presence of the third exogenouspolynucleotide encoding the OBC polypeptide, preferably a LDAP, togetherwith the first and second exogenous polynucleotides increases the totalnon-polar lipid content of the plant cell. preferably a cell invegetative plant part such as a leaf or stem, relative to acorresponding plant cell which comprises the first and second exogenouspolynucleotides but lacking the third exogenous polynucleotide. Morepreferably, the increase is synergistic. Most preferably, at least thepromoter that directs expression of the first exogenous polynucleotideis a promoter other than a constitutive promoter.

In a fourth aspect, the present invention provides a recombinanteukaryotic cell comprising plastids and a first exogenous polynucleotidewhich encodes a transcription factor polypeptide that increases theexpression of one or more glycolytic and/or fatty acid biosyntheticgenes in the cell, and one or more or all of;

a) a second exogenous polynucleotide which encodes a polypeptide whichincreases the export of fatty acids out of plastids of the cell whencompared to a corresponding cell lacking the second exogenouspolynucleotide,

b) a first genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in importing fattyacids into plastids of the cell when compared to a corresponding celllacking the first genetic modification, and

c) a second genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in diacylglycerol(DAG) production in the plastid when compared to a corresponding celllacking the second genetic modification, wherein each exogenouspolynucleotide is operably linked to a promoter which is capable ofdirecting expression of the polynucleotide in the cell.

In an embodiment, the cell, preferably a plant cell, comprises a) andoptionally b) or c).

In an embodiment, the cell of the above aspect further comprises one ormore or all of

d) a third exogenous polynucleotide which encodes a polypeptide involvedin the biosynthesis of one or more non-polar lipids,

e) a third genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in the catabolismof triacylglycerols (TAG) in the cell when compared to a correspondingcell lacking the third genetic modification, and

f) a fourth exogenous polynucleotide which encodes an oil body coating(OBC) polypeptide, preferably an LDAP.

In a preferred embodiment, the cell, preferably a plant cell, comprisesthe first, second and third exogenous polynucleotides and optionally thethird genetic modification or the fourth exogenous polynucleotide.

In a preferred embodiment, the presence of the second exogenouspolynucleotide encoding a polypeptide which increases the export offatty acids out of plastids of the cell, which is preferably a fattyacyl thioesterase such as a FATA polypeptide, together with the firstand, if present, third exogenous polynucleotides increases the totalnon-polar lipid content of the plant cell, preferably a cell invegetative plant part such as a leaf or stem, relative to acorresponding plant cell which comprises the first and, if present,third exogenous polynucleotides but lacking the second exogenouspolynucleotide. More preferably, the increase provided by the secondexogenous polynucleotide is synergistic. Most preferably, at least thepromoter that directs expression of the first exogenous polynucleotideis a promoter other than a constitutive promoter.

In an embodiment, the transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the cell is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1polypeptide or a LEC1-like polypeptide, preferably a transcriptionfactor other than Arabidopsis thaliana WRI1 (SEQ ID NOs:21 or 22), andthe polypeptide which increases the export of fatty acids out ofplastids of the cell is a fatty acid thioesterase, preferably a FATA ora FATB polypeptide, more preferably a FATA polypeptide or a fatty acidthioesterase other than a medium chain fatty acid thioesterase. Thepresence of a thioesterase other than a medium chain thioesterase isindicated by the percentage of C12:0 and/or C14:0 fatty acids in thetotal fatty acid content of the cell being about the same relative to acorresponding cell lacking the exogenous polynucleotide encoding thethioesterase. Preferably, the cell further comprises an exogenouspolynucleotide which encodes a DGAT and a genetic modification whichdown-regulates production of an endogenous SDP1 lipase. In anembodiment, the decreased production of an SDP1 lipase actssynergistically with the transcription factor and fatty acidthioesterase to increase the total non-polar lipid content in the cell.More preferably, the cell does not comprise an exogenous polynucleotideencoding a PDAT, and/or is a cell other than a Nicotiana benthamianacell. Most preferably, at least one of the exogenous polynucleotides inthe cell is expressed from a promoter which is not a constitutivepromoter such as, for example, a promoter expressed preferentially ingreen tissues or stems of the plant or that is up-regulated duringsenescence.

In an embodiment, the transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the cell is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1polypeptide or a LEC1-like polypeptide, and the polypeptide involved inimporting fatty acids into plastids of the cell is a TGD polypeptide.

In an embodiment, the transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the cell is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1polypeptide or a LEC1-like polypeptide, and the polypeptide involved indiacylglycerol (DAG) production is a plastidial GPAT.

In an embodiment, the transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the cell is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1polypeptide or a LEC1-like polypeptide, the polypeptide which increasesthe export of fatty acids out of plastids of the cell is a fatty acidthioesterase, preferably a FATA or a FATB polypeptide, and thepolypeptide involved in importing fatty acids into plastids of the cella TGD polypeptide.

In an embodiment, the transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the cell is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1polypeptide or a LEC1-like polypeptide, the polypeptide which increasesthe export of fatty acids out of plastids of the cell is a fatty acidthioesterase, preferably a FATA or a FATB polypeptide, and thepolypeptide involved in diacylglycerol (DAG) production is a plastidialGPAT.

In an embodiment, the transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the cell is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1polypeptide or a LEC1-like polypeptide, the polypeptide involved inimporting fatty acids into plastids of the cell a TGD polypeptide, andthe polypeptide involved in diacylglycerol (DAG) production is aplastidial GPAT.

In an embodiment, the cell comprises two exogenous polynucleotidesencoding two different transcription factor polypeptides that increasethe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the cell, such as WRI1 and LEC2, or WRI1 and LEC1.

In embodiments of the second, third and fourth aspects, when the cellcomprises an exogenous polynucleotide encoding a fatty acid thioesterasesuch as, for example, a FATA or a FATB polypeptide, the thioesterase ispreferably a FATA polypeptide or a fatty acid thioesterase other than amedium chain fatty acid thioesterase.

In a fifth aspect, the present invention provides a recombinanteukaryotic cell comprising

a) a first exogenous polynucleotide which encodes a transcription factorpolypeptide that increases the expression of one or more glycolyticand/or fatty acid biosynthetic genes in the cell, preferably a WRItranscription factor,

b) a second exogenous polynucleotide which encodes a polypeptideinvolved in the biosynthesis of one or more non-polar lipids which is anLPAAT with preferential activity for fatty acids with a medium chainlength (C8 to C14), and

c) a third exogenous polynucleotide which encodes a polypeptide whichincreases the export of C8 to C14 fatty acids out of plastids of thecell when compared to a corresponding cell lacking the third exogenouspolynucleotide,

wherein each exogenous polynucleotide is operably linked to a promoterwhich is capable of directing expression of the polynucleotide in thecell.

In an embodiment, the third exogenous polynucleotide encodes athioesterase, preferably a FATB thioesterase with preferential activityfor fatty acids with a medium chain length (C8 to C14).

In a preferred embodiment, the presence of the third exogenouspolynucleotide encoding a polypeptide which increases the export of C8to C14 fatty acids out of plastids of the cell, together with the firstand second exogenous polynucleotides increases the total MCFA content ofthe cell, preferably a cell in vegetative plant part such as a leaf,root or stem, relative to a corresponding plant cell which comprises thefirst and second exogenous polynucleotides but lacking the thirdexogenous polynucleotide. More preferably, the increase provided by thethird exogenous polynucleotide is synergistic. Most preferably, at leastthe promoter that directs expression of the first exogenouspolynucleotide is a promoter other than a constitutive promoter.

In an embodiment, the exogenous polynucleotide encoding the FATBthioesterase with preferential activity for fatty acids with a mediumchain length (C8 to C14) comprises amino acids whose sequence is setforth as any one of SEQ ID NOs:193 to 199, or a biologically activefragment of any one thereof, or a polypeptide whose amino acid sequenceis at least 30% identical to any one or more of SEQ ID NOs: 193 to 199.More preferably, the exogenous polynucleotide encoding the FATBthioesterase with preferential activity for fatty acids with a mediumchain length (C8 to C14) comprises amino acids whose sequence is setforth as SEQ ID NOs: 193 to 199, or a biologically active fragment ofany one thereof, or a polypeptide whose amino acid sequence is at least30% identical to any one or both of SEQ ID NOs: 193 to 199.

In an embodiment of the fifth aspect, the transcription factor is notArabidopsis thaliana WRI1 (SEQ ID NOs:21 or 22).

In an embodiment of the fifth aspect, the exogenous polynucleotideencoding LPAAT comprises amino acids whose sequence is set forth as SEQID NO:200, or a biologically active fragment thereof, or a polypeptidewhose amino acid sequence is at least 30% identical thereto.

In an embodiment of the fifth aspect, the cell further comprises one ormore or all of;

d) a fourth exogenous polynucleotide which encodes a further polypeptideinvolved in the biosynthesis of one or more non-polar lipids,

e) a first genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in the catabolismof triacylglycerols (TAG) in the cell when compared to a correspondingcell lacking the first genetic modification,

f) a fifth exogenous polynucleotide which encodes an oil body coating(OBC) polypeptide, preferably an LDAP,

g) a second genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in importing fattyacids into plastids of the cell when compared to a corresponding celllacking the second genetic modification, and

h) a third genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in diacylglycerol(DAG) production in the plastid when compared to a corresponding celllacking the third genetic modification, wherein each exogenouspolynucleotide is operably linked to a promoter which is capable ofdirecting expression of the polynucleotide in the cell.

In an embodiment of the fifth aspect, the cell is a plant cell from orin a vegetative part of a plant and one or more or all of the promotersare expressed at a higher level in the vegetative part relative to seedof the plant.

In an embodiment of the fifth aspect, the fatty acid with a medium chainlength is at least myristic acid. In a preferred embodiment, the cellcomprises a myristic acid content of at least about 8%, at least about10%, at least about 11%, at least about 12%, at least about 15%, atleast about 20%, at least about 25%, between 8% and 25%, between 8% and20%, between 10% and 25%, between 11% and 25%, between about 15% and25%, between about 20% and 25%, (w/w dry weight).

In the embodiments of the third, fourth and fifth aspects, it ispreferred that the cell is in a vegetative part of a plant which isgrowing in soil or which was grown in soil and the plant part wassubsequently harvested, and wherein the cell comprises at least 8% TAGon a weight basis (% dry weight) such as for example between 8% and 75%or between 8% and 30%. More preferably, the TAG content is at least 10%,such as for example between 10% and 75% or between 10% and 30%.Preferably, these TAG levels are present in the vegetative parts priorto or at flowering of the plant or prior to seed setting stage of plantdevelopment. In these embodiments, it is preferred that the ratio of theTAG content in the leaves to the TAG content in the stems of the plantis between 1:1 and 10:1, and/or the ratio is increased relative to acorresponding cell comprising the first and second exogenouspolynucleotides and lacking the first genetic modification.

In the embodiments of the second, third, fourth and fifth aspects, thecell preferably comprises an exogenous polynucleotide which encodes aDGAT and a genetic modification which down-regulates production of anendogenous SDP1 lipase. In a preferred embodiment, the cell does notcomprise an exogenous polynucleotide encoding a PDAT, and/or is a cellother than a Nicotiana benthamiana cell and/or is a cell other than aBrassica napus cell. Most preferably, at least one of the exogenouspolynucleotides in the cell is expressed from a promoter which is not aconstitutive promoter such as, for example, a promoter expressedpreferentially in green tissues or stems of the plant or that isup-regulated during senescence.

In an embodiment, a cell of the invention (including of the second,third, fourth and fifth aspects) has one or more or all of the followingfeatures (where applicable);

i) the cell has an increased synthesis of total fatty acids relative toa corresponding cell lacking the first exogenous polynucleotide, or adecreased catabolism of total fatty acids relative to a correspondingcell lacking the first exogenous polynucleotide, or both, such that ithas an increased level of total fatty acids relative to a correspondingcell lacking the first exogenous polynucleotide,

ii) the cell has an increased expression and/or activity of a fatty acylacyltransferase which catalyses the synthesis of TAG, DAG or MAG,preferably TAG, relative to a corresponding cell having the firstexogenous polynucleotide and lacking the exogenous polynucleotide whichencodes a polypeptide involved in the biosynthesis of one or morenon-polar lipids,

iii) the cell has a decreased production of lysophosphatidic acid (LPA)from acyl-ACP and G3P in its plastids relative to a corresponding cellhaving the first exogenous polynucleotide and lacking the geneticmodification which down-regulates endogenous production and/or activityof a polypeptide involved in diacylglycerol (DAG) production in theplastid in the cell,

iv) the cell has an altered ratio of C16:3 to C18:3 fatty acids in itstotal fatty acid content and/or its galactolipid content relative to acorresponding cell lacking the exogenous polynucleotide(s) and/orgenetic modification(s), preferably a decreased ratio,

v) the cell is in a vegetative part of a plant and comprises a totalnon-polar lipid content of at least about 8%, at least about 10%, atleast about 11%, at least about 12%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, between8% and 75%, between 10% and 75%, between 11% and 75%, between about 15%and 75%, between about 20% and 75%, between about 30% and 75%, betweenabout 40% and 75%, between about 50% and 75%, between about 60% and 75%,or between about 25% and 50% (w/w dry weight),

vi) the cell is in a vegetative part of a plant and comprises a TAGcontent of at least about 8%, at least about 10%, at least about 11%, atleast about 12%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, between 8% and 75%, between10% and 75%, between 11% and 75%, between about 15% and 75%, betweenabout 20% and 75%, between about 30% and 75%, between about 40% and 75%,between about 50% and 75%, between about 60% and 75%, or between about25% and 50% (w/w dry weight),

vii) the transcription factor polypeptide(s) is selected from the groupconsisting of Wrinkled 1 (WRI1), Leafy Cotyledon 1 (LEC1), LEC1-like,Leafy Cotyledon 2 (LEC2), BABY BOOM (BBM), FUS3, ABI3, ABI4, ABI5, Dof4and Dof11, or the group consisting of MYB73, bZIP53, AGL15, MYB115,MYB118, TANMEI, WUS, GFR2a1, GFR2a2 and PHR1,

viii) oleic acid comprises at least 20% (mol %), at least 22% (mol %),at least 30% (mol %), at least 40% (mol %), at least 50% (mol %), or atleast 60% (mol %), preferably about 65% (mol %) or between 20% and about65% of the total fatty acid content in the cell,

ix) non-polar lipid in the cell comprises a fatty acid which comprises ahydroxyl group, an epoxy group, a cyclopropane group, a doublecarbon-carbon bond, a triple carbon-carbon bond, conjugated doublebonds, a branched chain such as a methylated or hydroxylated branchedchain, or a combination of two or more thereof, or any of two, three,four, five or six of the aforementioned groups, bonds or branchedchains,

x) non-polar lipid in the cell comprises one or more polyunsaturatedfatty acids selected from eicosadienoic acid (EDA), arachidonic acid(ARA), stearidonic acid (SDA), eicosatrienoic acid (ETE),eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA),docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), or acombination of two of more thereof,

xi) the cell is in a plant or part thereof, preferably a vegetativeplant part, or the cell is an algal cell such as a diatom(bacillariophytes), green algae (chlorophytes), blue-green algae(cyanophytes), golden-brown algae (chrysophytes), haptophytes, brownalgae or heterokont algae, or the cell is from or is an organismsuitable for fermentation such as a fungus,

xii) one or more or all of the promoters are selected from a promoterother than a constitutive promoter, preferably a tissue-specificpromoter such as a leaf and/or stem specific promoter, a developmentallyregulated promoter such as a senescense-specific promoter such as aSAG12 promoter, an inducible promoter, or a circadian-rhythm regulatedpromoter, preferably wherein at least one of the promoters operablylinked to an exogenous polynucleotide which encodes a transcriptionfactor polypeptide is a promoter other than a constitutive promoter,

xiii) the cell comprises a total fatty acid content which comprisesmedium chain fatty acids, preferably C12:0, C14:0 or both, at a level ofat least 5% of the total fatty acid content and optionally an exogenouspolynucleotide which encodes an LPAAT which has preferential activityfor fatty acids with a medium chain length (C8 to C14), preferably C12:0or C14:0,

xiv) the cell comprises a total fatty acid content whose oleic acidlevel and/or palmitic acid level is increased by at least 2% relative toa corresponding cell lacking the exogenous polynucleotide(s) and/orgenetic modification(s), and/or whose α-linolenic acid (ALA) leveland/or linoleic acid level is decreased by at least 2% relative to acorresponding cell lacking the exogenous polynucleotide(s) and/orgenetic modification(s),

xv) non-polar lipid in the cell comprises a modified level of totalsterols, preferably free (non-esterified) sterols, steroyl esters,steroyl glycosides, relative to the non-polar lipid in a correspondingcell lacking the exogenous polynucleotide(s) and/or geneticmodification(s),

xvi) non-polar lipid in the cell comprises waxes and/or wax esters,

xvii) the cell is one member of a population or collection of at leastabout 1000 such cells, preferably in a vegetative plant part or a seed,

xviii) the cell comprises an exogenous polynucleotide encoding asilencing suppressor, wherein the exogenous polynucleotide is operablylinked to a promoter which is capable of directing expression of thepolynucleotide in the cell,

xix) the level of one or more non-polar lipid(s) and/or the totalnon-polar lipid content of the cell is at least 2% greater on a weightbasis than in a corresponding cell which comprises exogenouspolynucleotides encoding an Arabidposis thaliana WRI1 (SEQ ID NO:21) andan Arabidopsis thaliana DGAT1 (SEQ ID NO: 1), and

xx) a total polyunsaturated fatty acid (PUFA) content which is decreasedrelative to the total PUFA content of a corresponding cell lacking theexogenous polynucleotide(s) and/or genetic modification(s).

The following embodiments apply to the cell of the invention (includingof the second, third, fourth and fifth aspects) as well as to methods ofproducing the cells and to methods of using the cells. In theseembodiments, where the cell is in a vegetative part of a plant, it ispreferred that the plant is growing in soil or was grown in soil.

In an embodiment, the polypeptide involved in the biosynthesis of one ormore non-polar lipids is a fatty acyl acyltransferase which is involvedin the biosynthesis of TAG, DAG or monoacylglycerol (MAG) in the cell,preferably of TAG in the cell, such as, for example, a DGAT, PDAT,LPAAT, GPAT or MGAT, preferably a DGAT or a PDAT.

In an embodiment, the polypeptide involved in the catabolism oftriacylglycerols (TAG) in the cell is an SDP1 lipase, a Cgi58polypeptide, an acyl-CoA oxidase such as ACX1 or ACX2, or a polypeptideinvolved in β-oxidation of fatty acids in the cell such as a PXA1peroxisomal ATP-binding cassette transporter, preferably an SDP1 lipase.

In an embodiment, the oil body coating (OBC) polypeptide is oleosin,such as a polyoleosin or a caleosin, or preferably a lipid dropletassociated protein (LDAP).

In an embodiment, the polypeptide which increases the export of fattyacids out of plastids of the cell is a C16 or C18 fatty acidthioesterase such as a FATA polypeptide or a FATB polypeptide, a fattyacid transporter such as an ABCA9 polypeptide or a long-chain acyl-CoAsynthetase (LACS).

In an embodiment, the polypeptide involved in importing fatty acids intoplastids of the cell is a fatty acid transporter, or subunit thereof,preferably a TGD polypeptide such as, for example, a TGD1 polypeptide, aTGD2 polypeptide, a TGD3 polypeptide, or a TGD4 polypeptide.

In an embodiment, the polypeptide involved in diacylglycerol (DAG)production in the plastid is a plastidial GPAT, a plastidial LPAAT or aplastidial PAP.

In one embodiment, the cell is from or in a 16:3 plant, or in avegetative part or seed thereof, and which comprises one or more or allof the following;

a) an exogenous polynucleotide which encodes a polypeptide whichincreases the export of fatty acids out of plastids of the cell whencompared to a corresponding cell lacking the exogenous polynucleotide,

b) a first genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in importing fattyacids into plastids of the cell when compared to a corresponding celllacking the first genetic modification, and

c) a second genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in diacylglycerol(DAG) production in the plastid when compared to a corresponding celllacking the second genetic modification, wherein the exogenouspolynucleotide is operably linked to a promoter which is capable ofdirecting expression of the polynucleotide in the cell.

In an alternative embodiment, the cell is from or in a 18:3 plant, or ina vegetative part or seed thereof.

In an embodiment, the cell is from or in a plant leaf, stem or root,before the plant flowers, and the cell comprises a total non-polar lipidcontent of at least about 8%, at least about 10%, at least about 11%,between 8% and 15%, or between 9% and 12% (w/w dry weight). In anembodiment, the total non-polar lipid content of the cell is at least3%, more preferably at least 5% greater, than the total non-polar lipidcontent in a corresponding cell transformed with genes encoding a WRI1and a DGAT but lacking the other exogenous polynucleotides and geneticmodifications as described herein for the second, third, fourth andfifth aspects. More preferably, that degree of increase is in a cell ina stem or root of the plant.

In an embodiment, the addition of one or more of the exogenouspolynucleotides or genetic modifications, preferably the exogenouspolynucleotide encoding an OBC or a fatty acyl thioesterase or thegenetic modification which down-regulates endogenous production and/oractivity of a polypeptide involved in the catabolism of triacylglycerols(TAG) in the cell, more preferably the exogenous polynucleotide whichencodes a FATA thioesterase or an LDAP or which decreases expression ofan endogenous TAG lipase such as a SDP1 TAG lipase in the cell, resultsin a synergistic increase in the total non-polar lipid content of thecell when added to the pair of transgenes WRI1 and DGAT, particularlybefore the plant flowers and even more particularly in the stems and/orroots of the plant. For example, see Examples 8, 11 and 15. In apreferred embodiment, the increase in the TAG content of the cell in astem or root of the plant is at least 2-fold, more preferably at least3-fold, relative to a corresponding cell transformed with genes encodingWRI1 and DGAT1 but lacking the FATA thioesterase, LDAP and the geneticmodification which down-regulates endogenous production and/or activityof a polypeptide involved in the catabolism of triacylglycerols (TAG) inthe cell. Most preferably, at least the promoter that directs expressionof the first exogenous polynucleotide is a promoter other than aconstitutive promoter.

The genetic modification can be any change to a naturally occurring cellthat achieves the desired effect. Methods of genetically modifying cellsare well known in the art. In an embodiment, each of the one or more orall of the genetic modifications is a mutation of an endogenous genewhich partially or completely inactivates the gene, preferably anintroduced mutation, such as a point mutation, an insertion, or adeletion (or a combination of one or more thereof). The point mutationmay be a premature stop codon, a splice-site mutation, a frame-shiftmutation or an amino acid substitution mutation that reduces activity ofthe gene or the encoded polypeptide. The deletion may be of one or morenucleotides within a transcribed exon or promoter of the gene, or extendacross or into more than one exon, or extend to deletion of the entiregene. Preferably the deletion is introduced by use of ZF, TALEN orCRISPR technologies. In an embodiment, one or more or all of the geneticmodifications is an exogenous polynucleotide encoding an RNA moleculewhich inhibits expression of the endogenous gene, wherein the exogenouspolynucleotide is operably linked to a promoter which is capable ofdirecting expression of the polynucleotide in the cell. Examples ofexogenous polynucleotide which reduces expression of an endogenous geneare selected from the group consisting of an antisense polynucleotide, asense polynucleotide, a microRNA, a polynucleotide which encodes apolypeptide which binds the endogenous enzyme, a double stranded RNAmolecule and a processed RNA molecule derived therefrom. In anembodiment, the cell comprises genetic modifications which are anintroduced mutation in an endogenous gene and an exogenouspolynucleotide encoding an RNA molecule which reduces expression ofanother endogenous gene.

In an embodiment, the exogenous polynucleotide encoding WRI1 comprisesone or more of the following:

i) nucleotides encoding a polypeptide comprising amino acids whosesequence is set forth as any one of SEQ ID NOs:21 to 75 or 205 to 210,or a biologically active fragment thereof, or a polypeptide whose aminoacid sequence is at least 30% identical to any one or more of SEQ IDNOs: 21 to 75 or 205 to 210,

ii) nucleotides whose sequence is at least 30% identical to i), and

iii) nucleotides which hybridize to i) and/or ii) under stringentconditions.

Preferably, the WRI1 polypeptide is a WRI1 polypeptide other thanArabidopsis thaliana WRI1 (SEQ ID NOs:21 or 22). More preferably, theWRI1 polypeptide comprises amino acids whose sequence is set forth asSEQ ID NO:208, or a biologically active fragment thereof, or apolypeptide whose amino acid sequence is at least 30% identical thereto.

In an embodiment of the second, third, fourth or fifth aspects, therecombinant cell is a cell of a potato (Solanum tuberosum) tuber, a cellof a sugarbeet (Beta vulgaris) beet or leaf, a cell of a sugarcane(Saccharum sp.) or sorghum (Sorghumn bicolor) stem or leaf, an endospermcell of a monocotyledonous plant, wherein the cell has an increasedtotal fatty acid content relative to a corresponding wild-type endospermcell such as, for example, a cell of a wheat (Triticum aestivum) grain,rice (Oryza sp.) grain or a corn (Zea mays) kernel, a cell of a Brassicasp. seed having an increased total fatty acid content such as, forexample, a canola seed, or a cell of a legume seed having an increasedtotal fatty acid content such as, for example, a soybean (Glycine max)seed.

In a sixth aspect, the present invention provides a non-human organism,or part thereof, comprising, or consisting of, one or more cells of theinvention.

In an embodiment, the part of the non-human organism is a seed, fruit,or a vegetative part of a plant such as an aerial plant part or a greenpart such as a leaf or stem.

In another embodiment, the non-human organism is a phototrophic organismsuch as, for example, a plant or an alga, or an organism suitable forfermentation such as, for example, a fungus.

In a seventh aspect, the present invention provides a transgenic plant,or part thereof, preferably a vegetative plant part, comprising

a) a first exogenous polynucleotide which encodes a transcription factorpolypeptide that increases the expression of one or more glycolyticand/or fatty acid biosynthetic genes in the plant,

b) a second exogenous polynucleotide which encodes a polypeptideinvolved in the biosynthesis of one or more non-polar lipids, and anyone or two or all three of

c) a genetic modification which down-regulates endogenous productionand/or activity of a polypeptide involved in the catabolism oftriacylglycerols (TAG) in the plant when compared to a correspondingplant lacking the genetic modification,

d) a third exogenous polynucleotide which encodes a polypeptide whichincreases the export of fatty acids out of plastids of cells of theplant when compared to a corresponding cell lacking the fourth exogenouspolynucleotide, and

e) a fourth exogenous polynucleotide which encodes a secondtranscription factor polypeptide that increases the expression of one ormore glycolytic and/or fatty acid biosynthetic genes in the plant,

wherein each exogenous polynucleotide is operably linked to a promoterwhich is capable of directing expression of the polynucleotide in theplant.

In an embodiment, the plant or part thereof comprises a), b) and c), andoptionally d) or e).

In an embodiment, the plant or part thereof comprises a), b) and d), andoptionally c) or e).

In an embodiment, the plant or part thereof comprises a), b) and e), andoptionally c) or d).

In a preferred embodiment, the presence of c), d) or e), together witha) and b) increases the total non-polar lipid content of the plant orpart thereof, preferably a vegetative plant part such as a leaf, root orstem, relative to a corresponding plant or part thereof which comprisesa) and b) but lacking each of c), d) and e). More preferably, theincrease is synergistic. Most preferably, at least the promoter thatdirects expression of the first exogenous polynucleotide is a promoterother than a constitutive promoter.

In an embodiment, the plant, or part thereof, further comprises one ormore or all of

a) a fifth exogenous polynucleotide which encodes an oil body coating(OBC) polypeptide, preferably an LDAP,

b) a second genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in importing fattyacids into plastids of the plant when compared to a corresponding plantlacking the second genetic modification, and

c) a third genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in diacylglycerol(DAG) production in the plastid when compared to a corresponding plantlacking the third genetic modification.

In an embodiment, the transgenic plant, or part thereof, comprises

a) a first exogenous polynucleotide which encodes a transcription factorpolypeptide that increases the expression of one or more glycolyticand/or fatty acid biosynthetic genes in the plant, preferably expressedfrom a promoter other than a constitutive promoter,

b) a second exogenous polynucleotide which encodes a polypeptideinvolved in the biosynthesis of one or more non-polar lipids, and

c) a genetic modification which down-regulates endogenous productionand/or activity of a polypeptide involved in the catabolism oftriacylglycerols (TAG) in the plant when compared to a correspondingplant lacking the genetic modification,

wherein each exogenous polynucleotide is operably linked to a promoterwhich is capable of directing expression of the polynucleotide in theplant, and optionally the plant, or part thereof, further comprises oneor more or all of

d) a third exogenous polynucleotide which encodes an oil body coating(OBC) polypeptide, preferably an LDAP,

e) a fourth exogenous polynucleotide which encodes a polypeptide whichincreases the export of fatty acids out of plastids of the plant whencompared to a corresponding plant lacking the fourth exogenouspolynucleotide,

f) a second genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in importing fattyacids into plastids of the plant when compared to a corresponding plantlacking the second genetic modification, and

g) a third genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in diacylglycerol(DAG) production in the plastid when compared to a corresponding plantlacking the third genetic modification.

In an embodiment, the part is a vegetative part and one or more or allof the promoters are expressed at a higher level in the vegetative partrelative to seed of the plant.

In an embodiment, the polypeptide involved in the biosynthesis of one ormore non-polar lipids is a DGAT or a PDAT and the polypeptide involvedin the catabolism of TAG in the plant is an SDP1 lipase.

In an embodiment, the transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the plant is a WRI1 polypeptide and the polypeptide involved inthe biosynthesis of one or more non-polar lipids is a DGAT or a PDAT.

In an embodiment, the transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the plant is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1polypeptide or a LEC1-like polypeptide and the polypeptide involved inthe biosynthesis of one or more non-polar lipids is a DGAT.

In an embodiment, the transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the plant is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1polypeptide or a LEC1-like polypeptide and the polypeptide involved inthe catabolism of triacylglycerols (TAG) in the plant is an SDP1 lipase.

In an embodiment, the transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the plant is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1polypeptide or a LEC1-like polypeptide, the polypeptide involved in thebiosynthesis of one or more non-polar lipids is a DGAT or a PDAT and thepolypeptide involved in the catabolism of triacylglycerols (TAG) in theplant is an SDP1 lipase.

In an embodiment, when present, the two transcription factors are WRI1and LEC2, or WRI1 and LEC1.

In the above embodiments, it is preferred that the plant is growing insoil or was grown in soil and the part thereof was subsequentlyharvested. Preferably, a vegetative part of the plant comprises at least8% TAG on a weight basis (% dry weight) such as for example between 8%and 75% or between 8% and 30%. More preferably, the TAG content is atleast 10%, such as for example between 10% and 75% or between 10% and30%. Preferably, these TAG levels are present in the vegetative partprior to or at flowering of the plant or prior to seed setting stage ofplant development. In these embodiments, it is preferred that the ratioof the TAG content in the leaves to the TAG content in the stems of theplant is between 1:1 and 10:1, and/or the ratio is increased relative toa corresponding cell comprising the first and second exogenouspolynucleotides and lacking the first genetic modification.

In the above embodiments, the total non-polar lipid content of the plantor part thereof is preferably at least 3%, more preferably at least 5%greater, than the total non-polar lipid content in a corresponding plantor part thereof transformed with genes encoding a WRI1 and a DGAT butlacking the other exogenous polynucleotides and genetic modifications asdescribed herein. More preferably, that degree of increase is in stem orroot tissues of the plant.

In the above embodiments, it is preferred that the addition of one ormore exogenous polynucleotides or genetic modifications, preferably theexogenous polynucleotide encoding the OBC or the fatty acid thioesteraseor the genetic modification which down-regulates endogenous productionand/or activity of a polypeptide involved in the catabolism oftriacylglycerols (TAG) in the cell, more preferably the exogenouspolynucleotide which encodes an LDAP or FATA thioesterase or whichdecreases expression of an endogenous TAG lipase such as a SDP1 TAGlipase in the cell, results in a synergistic increase in the totalnon-polar lipid content of the plant or part thereof when added to thepair of transgenes WRI1 and DGAT, particularly before the plant flowersand even more particularly in stem and/or root tissue of the plant. Forexample, see Examples 8, 11 and 15. In a preferred embodiment, theincrease in the TAG content of the leaf, stem or root tissues, or allthree, of the plant is at least 2-fold, more preferably at least 3-fold,relative to a corresponding part transformed with genes encoding WRI1and DGAT1 but lacking the exogenous polynucleotide encoding the OBC orthe fatty acid thioesterase and the genetic modification whichdown-regulates endogenous production and/or activity of a polypeptideinvolved in the catabolism of triacylglycerols (TAG) in the cell.

In the above embodiments, the plant or part thereof preferably comprisesa second exogenous polynucleotide which encodes a DGAT and a firstgenetic modification which down-regulates production of an endogenousSDP1 lipase. More preferably, the plant or part thereof does notcomprise an exogenous polynucleotide encoding a PDAT, and/or is a plantor part thereof other than of Nicotiana benthamiana and/or Brassicanapus, and/or the WRI1 is a WRI1 other than Arabidopsis thaliana WRI1(SEQ ID NOs:21 or 22). In an embodiment, the plant is other thansugarcane. Most preferably, at least one of the exogenouspolynucleotides in the plant is expressed from a promoter which is not aconstitutive promoter such as, for example, a promoter which isexpressed preferentially in green tissues or stems of the plant or thatis up-regulated after commencement of flowering or during senescence.Preferably at least the first exogenous polynucleotide (encoding atranscription factor) is expressed from such a promoter.

In an eighth aspect, the present invention provides a transgenic plant,or part thereof, preferably a vegetative plant part, comprising

a) a first exogenous polynucleotide which encodes a transcription factorpolypeptide that increases the expression of one or more glycolyticand/or fatty acid biosynthetic genes in the plant,

b) a second exogenous polynucleotide which encodes a polypeptideinvolved in the biosynthesis of one or more non-polar lipids, and

c) a third exogenous polynucleotide which encodes an oil body coating(OBC) polypeptide, preferably an LDAP,

wherein each exogenous polynucleotide is operably linked to a promoterwhich is capable of directing expression of the polynucleotide in theplant and wherein the plant has an increased level of one or morenon-polar lipid(s) and/or an increased amount of the OBC polypeptide,relative to a corresponding plant which comprises a third exogenouspolynucleotide whose nucleotide sequence is the complement of thesequence provided as SEQ ID NO:176.

In a preferred embodiment, the presence of the third exogenouspolynucleotide encoding the OBC polypeptide, together with the first andsecond exogenous polynucleotides, increases the total non-polar lipidcontent of the plant or part thereof, preferably a vegetative plant partsuch as a leaf, root or stem, relative to a corresponding plant partwhich comprises the first and second exogenous polynucleotides butlacking the third exogenous polynucleotide. More preferably, theincrease is synergistic. Most preferably, at least the promoter thatdirects expression of the first exogenous polynucleotide is a promoterother than a constitutive promoter.

In an embodiment of the eighth aspect, the plant, or part thereof,further comprises one or more or all of

d) a first genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in the catabolismof triacylglycerols (TAG) in the plant when compared to a correspondingplant lacking the first genetic modification,

e) a fourth exogenous polynucleotide which encodes a polypeptide whichincreases the export of fatty acids out of plastids of the plant whencompared to a corresponding plant lacking the fourth exogenouspolynucleotide,

f) a second genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in importing fattyacids into plastids of the plant when compared to a corresponding plantlacking the second genetic modification, and

g) a third genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in diacylglycerol(DAG) production in the plastid when compared to a corresponding plantlacking the third genetic modification.

In an embodiment, the polypeptide involved in the biosynthesis of one ormore non-polar lipids is a DGAT or a PDAT and the OBC polypeptide is anoleosin. Alternatively, the OBC polypeptide is an LDAP.

In an embodiment, the transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the plant is a WRI1 polypeptide and the polypeptide involved inthe biosynthesis of one or more non-polar lipids is a DGAT or a PDAT.

In an embodiment, the transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the plant is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1polypeptide or a LEC1-like polypeptide and the OBC polypeptide is anoleosin. Alternatively, the OBC polypeptide is an LDAP.

In an embodiment, the transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the plant is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1polypeptide or a LEC1-like polypeptide, the polypeptide involved in thebiosynthesis of one or more non-polar lipids is a DGAT or a PDAT and theOBC polypeptide is an oleosin. Alternatively, the OBC polypeptide is anLDAP.

In an embodiment, the cell comprises two exogenous polynucleotidesencoding two different transcription factor polypeptides that increasethe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the plant, such as WRI1 and LEC2, or WRI1 and LEC1.

In an ninth aspect, the present invention provides a transgenic plant orpart thereof, preferably a vegetative plant part, comprising a firstexogenous polynucleotide which encodes a transcription factorpolypeptide that increases the expression of one or more glycolyticand/or fatty acid biosynthetic genes in the plant and one or more or allof;

a) a second exogenous polynucleotide which encodes a polypeptide whichincreases the export of fatty acids out of plastids of the plant whencompared to a corresponding plant lacking the second exogenouspolynucleotide,

b) a first genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in importing fattyacids into plastids of the plant when compared to a corresponding plantlacking the first genetic modification, and

c) a second genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in diacylglycerol(DAG) production in the plastid when compared to a corresponding plantlacking the second genetic modification,

wherein each exogenous polynucleotide is operably linked to a promoterwhich is capable of directing expression of the polynucleotide in theplant.

In an embodiment, the plant or part thereof, preferably a vegetativeplant part, comprises a) and optionally b) or c).

In an embodiment of the ninth aspect, the plant, or part thereof,further comprises one or more or all of

d) a third exogenous polynucleotide which encodes a polypeptide involvedin the biosynthesis of one or more non-polar lipids,

e) a third genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in the catabolismof triacylglycerols (TAG) in the plant when compared to a correspondingplant lacking the third genetic modification, and

f) a fourth exogenous polynucleotide which encodes an oil body coating(OBC) polypeptide, preferably an LDAP.

In a preferred embodiment, the plant or part thereof, preferably avegetative plant part, comprises the first, second and third exogenouspolynucleotides and optionally the third genetic modification or thefourth exogenous polynucleotide.

In a preferred embodiment, the presence of the second exogenouspolynucleotide encoding a polypeptide which increases the export offatty acids out of plastids of the plant, which is preferably a fattyacyl thioesterase such as a FATA polypeptide, together with the firstand, if present, third exogenous polynucleotides increases the totalnon-polar lipid content of the plant part, preferably a vegetative plantpart such as a leaf, root or stem, relative to a corresponding plantpart which comprises the first and, if present, third exogenouspolynucleotides but lacking the second exogenous polynucleotide. Morepreferably, the increase provided by the second exogenous polynucleotideis synergistic. Most preferably, at least the promoter that directsexpression of the first exogenous polynucleotide is a promoter otherthan a constitutive promoter.

In an embodiment, the transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the plant is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1polypeptide or a LEC1-like polypeptide, and the polypeptide involved inimporting fatty acids into plastids of the cell is a TGD polypeptide.

In an embodiment, the transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the plant is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1polypeptide or a LEC1-like polypeptide, and the polypeptide involved indiacylglycerol (DAG) production is a plastidial GPAT.

In an embodiment, the transcription, factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the plant is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1polypeptide or a LEC1-like polypeptide, the polypeptide which increasesthe export of fatty acids out of plastids of the plant is a fatty acidthioesterase, preferably a FATA or a FATB polypeptide, and thepolypeptide involved in importing fatty acids into plastids of the plantis a TGD polypeptide.

In an embodiment, the transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the plant is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1polypeptide or a LEC1-like polypeptide, the polypeptide which increasesthe export of fatty acids out of plastids of the plant is a fatty acidthioesterase, preferably a FATA or a FATB polypeptide, and thepolypeptide involved in diacylglycerol (DAG) production is a plastidialGPAT.

In an embodiment, the transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the plant is a WRI1 polypeptide, a LEC2 polypeptide, a LEC1polypeptide or a LEC1-like polypeptide, the polypeptide involved inimporting fatty acids into plastids of the plant a TGD polypeptide, andthe polypeptide involved in diacylglycerol (DAG) production is aplastidial GPAT.

In an embodiment, the plant comprises two exogenous polynucleotidesencoding two different transcription factor polypeptides that increasethe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the cell, such as WRI1 and LEC2, or WRI1 and LEC1.

In embodiments of the seventh, eighth and ninth aspects, when the plantcomprises an exogenous polynucleotide encoding a fatty acid thioesterasesuch as, for example, a FATA or a FATB polypeptide, the thioesterase ispreferably a FATA polypeptide or a fatty acid thioesterase other than amedium chain fatty acid thioesterase.

In a tenth aspect, the present invention provides a transgenic plant, orpart thereof, preferably a vegetative part, comprising

a) a first exogenous polynucleotide which encodes a transcription factorpolypeptide that increases the expression of one or more glycolyticand/or fatty acid biosynthetic genes in the plant, preferably a WRI1transcription factor,

b) a second exogenous polynucleotide which encodes a polypeptideinvolved in the biosynthesis of one or more non-polar lipids which is anLPAAT with preferential activity for fatty acids with a medium chainlength (C8 to C14), and

c) a third exogenous polynucleotide which encodes a polypeptide whichincreases the export of C8 to C14 fatty acids out of plastids of theplant when compared to a corresponding a plant lacking the thirdexogenous polynucleotide,

wherein each exogenous polynucleotide is operably linked to a promoterwhich is capable of directing expression of the polynucleotide in theplant.

In a preferred embodiment, the presence of the third exogenouspolynucleotide encoding a polypeptide which increases the export of C8to C14 fatty acids out of plastids of the plant, together with the firstand second exogenous polynucleotides increases the total MCFA content ofthe plant part, preferably a vegetative plant part such as a leaf, rootor stem, relative to a corresponding plant part which comprises thefirst and second exogenous polynucleotides but lacking the thirdexogenous polynucleotide. More preferably, the increase provided by thethird exogenous polynucleotide is synergistic. Most preferably, at leastthe promoter that directs expression of the first exogenouspolynucleotide is a promoter other than a constitutive promoter.

In an embodiment of the tenth aspect, the transgenic plant or partthereof further comprises one or more or all of;

d) a fourth exogenous polynucleotide which encodes a further polypeptideinvolved in the biosynthesis of one or more non-polar lipids,

e) a first genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in the catabolismof triacylglycerols (TAG) in the plant when compared to a correspondingplant lacking the first genetic modification,

f) a fifth exogenous polynucleotide which encodes an oil body coating(OBC) polypeptide, preferably an LDAP,

g) a second genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in importing fattyacids into plastids of the plant when compared to a corresponding plantlacking the second genetic modification, and

h) a third genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in diacylglycerol(DAG) production in the plastid when compared to a corresponding plantlacking the third genetic modification,

wherein each exogenous polynucleotide is operably linked to a promoterwhich is capable of directing expression of the polynucleotide in theplant.

In an embodiment of the tenth aspect, the transcription factor is notArabidopsis thaliana WRI1 (SEQ ID NOs:21 or 22), and/or the plant is notN. benthamiana.

In an embodiment of the tenth aspect, the exogenous polynucleotideencoding LPAAT comprises amino acids whose sequence is set forth as SEQID NO:200, or a biologically active fragment thereof, or a LPAATpolypeptide whose amino acid sequence is at least 30% identical thereto.

In an embodiment of the tenth aspect, one or more or all of thepromoters are expressed at a higher level in the vegetative partrelative to seed of the plant, preferably including at least thepromoter that expresses the first exogenous polynucleotide.

In an embodiment of the tenth aspect, the fatty acid with a medium chainlength is at least myristic acid (C14:0). In a preferred embodiment, theplant part, preferably a vegetative plant part, comprises a myristicacid content of at least about 8%, at least about 10%, at least about11%, at least about 12%, at least about 15%, at least about 20%, atleast about 25%, between 8% and 25%, between 8% and 20%, between 10% and25%, between 11% and 25%, between about 15% and 25%, between about 20%and 25%, (w/w dry weight).

In the embodiments of the sixth, seventh, eighth, ninth and tenthaspects, it is preferred that the plant is growing in soil or was grownin soil and the plant part, preferably vegetative plant part, wassubsequently harvested, and wherein the plant part comprises at least 8%TAG on a weight basis (% dry weight) such as for example between 8% and75% or between 8% and 30%. More preferably, the TAG content is at least10%, such as for example between 10% and 75% or between 10% and 30%.Preferably, these TAG levels are present in the vegetative part prior toor at flowering of the plant or prior to seed setting stage of plantdevelopment. In these embodiments, it is preferred that the ratio of theTAG content in the leaves to the TAG content in the stems of the plantis between 1:1 and 10:1, and/or the ratio is increased relative to acorresponding cell comprising the first and second exogenouspolynucleotides and lacking the first genetic modification.

In the embodiments of the sixth, seventh, eighth, ninth and tenthaspects, the plant or part thereof preferably comprises an exogenouspolynucleotide which encodes a DGAT and a genetic modification whichdown-regulates production of an endogenous SDP1 lipase. In a preferredembodiment, the plant or part thereof does not comprise an exogenouspolynucleotide encoding a PDAT, and/or is a plant other than a Nicotianabenthamiana plant. Most preferably, at least one of the exogenouspolynucleotides in the plant or part thereof is expressed from apromoter which is not a constitutive promoter such as, for example, apromoter expressed preferentially in green tissues or stems of the plantor that is up-regulated during senescence.

In an eleventh aspect, the present invention provides a plant comprisinga vegetative part, or the vegetative part thereof, wherein thevegetative part has a total non-polar lipid content of at least about18%, at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 55%, at least about 60%, at least about 65%, atleast about 70%, between 18% and 75%, between about 20% and 75%, betweenabout 30% and 75%, between about 40% and 75%, between about 50% and 75%,between about 60% and 75%, or between about 25% and 50% (w/w dryweight), wherein the non-polar lipid comprises at least 90%triacylglycerols (TAG).

In preferred embodiments, the vegetative plant part is characterised byfeatures as described in the seventh, eighth, ninth and tenth aspects.The plant is preferably an 18:3 plant.

In an embodiment of the above aspects, the plant cell or plant part hasbeen treated so it is no longer able to be propagated or give rise to aliving plant, i.e. it is dead. For example, the plant cell or plant parthas been dried and/or ground.

In an twelfth aspect, the present invention provides a plant comprisinga vegetative part, or the vegetative part thereof, wherein thevegetative part has a TAG content of at least about 18%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, between18% and 75%, between about 20% and 75%, between about 30% and 75%,between about 40% and 75%, between about 50% and 75%, between about 60%and 75%, or between about 25% and 50% (w/w dry weight), wherein thenon-polar lipid comprises at least 90% triacylglycerols (TAG). The plantis preferably an 18:3 plant.

In a thirteenth aspect, the present invention provides a plantcomprising a vegetative part, or the vegetative part thereof, whereinthe vegetative part has a total non-polar lipid content of at least 8%,at least about 10%, at least about 1%, at least about 12%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, between 8% and 75%, between 10% and 75%, between 11%and 75%, between about 15% and 75%, between about 20% and 75%, betweenabout 30% and 75%, between about 40% and 0.75%, between about 50% and75%, between about 60% and 75%, or between about 25% and 50% (w/w dryweight), wherein the non-polar lipid comprises at least 90%triacylglycerols (TAG), and wherein the plant is a 16:3 plant orvegetative part thereof.

In a fourteenth aspect, the present invention provides a plantcomprising a vegetative part, or the vegetative part thereof, whereinthe vegetative part has a TAG content of at least 8%, at least about10%, at least about 11%, at least about 12%, at least about 15%, atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, between 8% and 75%, between 10% and 75%, between 11% and 75%,between about 15% and 75%, between about 20% and 75%, between about 30%and 75%, between about 40% and 75%, between about 50% and 75%, betweenabout 60% and 75%, or between about 25% and 50% (w/w dry weight),wherein the non-polar lipid comprises at least 90% triacylglycerols(TAG), and wherein the plant is a 16:3 plant or vegetative part thereof.

In an embodiment, the cell of the invention (including of the second,third, fourth and fifth aspects) is a cell of the following species orgenera, or the plant or part thereof of the invention (including of thesixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth andfourteenth aspects) is Acrocomia aculeata (macauba palm), Arabidopsisthaliana, Aracinis hypogaea (peanut), Astrocaryum murumuru (murumuru),Astrocaryum vulgare (tucumã), Attalea geraensis (Indaiá-rateiro),Attalea humilis (American oil palm), Attalea oleifera (andaiá), Attaleaphalerata (uricuri), Attalea speciosa (babassu), Avena sativa (oats),Beta vulgaris (sugar beet), Brassica sp. such as, for example, Brassicacarinata, Brassica juncea, Brassica napobrassica, Brassica napus(canola), Camelina sativa (false flax), Cannabis sativa (hemp),Carthamus tinctorius (safflower), Caryocar brasiliense (pequi), Cocosnucifera (Coconut), Crambe abyssinica (Abyssinian kale), Cucumis melo(melon), Elaeis guineensis (African palm), Glycine max (soybean),Gossypium hirsutum (cotton), Helianthus sp. such as Helianthus annuus(sunflower), Hordeum vulgare (barley), Jatropha curcas (physic nut),Joannesia princeps (arara nut-tree), Lemna sp. (duckweed) such as Lemnaaequinoctialis, Lemna disperma, Lemna ecuadoriensis, Lemna gibba(swollen duckweed), Lemna japonica, Lemna minor, Lemna minuta, Lemnaobscura, Lemna paucicostata, Lemna perpusilla, Lemna tenera, Lemnatrisulca, Lemna turionifera, Lemna valdiviana, Lemna yungensis, Licaniarigida (oiticica), Linum usitatissimum (flax), Lupinus angustifolius(lupin), Mauritia flexuosa (buriti palm), Maximiliana maripa (inajapalm), Miscanthus sp. such as Miscanthus x giganteus and Miscanthussinensis, Nicotiana sp. (tabacco) such as Nicotiana tabacum or Nicotianabenthamiana, Oenocarpus bacaba (bacaba-do-azeite), Oenocarpus bataua(patauã), Oenocarpus distichus (bacaba-de-leque), Oryza sp. (rice) suchas Oryza sativa and Oryza glaberrima, Panicum virgatum (switchgrass),Paraqueiba paraensis (mari), Persea amencana (avocado), Pongamia pinnata(Indian beech), Populus trichocarpa, Ricinus communis (castor),Saccharum sp. (sugarcane), Sesamum indicum (sesame), Solanum tuberosum(potato), Sorghum sp. such as Sorghum bicolor, Sorghum vulgare,Theobroma grandiforum (cupuassu), Trifolium sp., Trithrinax brasiliensis(Brazilian needle palm), Triticum sp. (wheat) such as Triticum aestivumand Zea mays (corn).

In a fifteenth aspect, the present invention provides a potato plant, orpart thereof preferably a tuber which has a diameter of at least 2 cm,and has a TAG content of at least 0.5% on a dry weight basis and/or atotal fatty acid content of at least 1%, preferably at least 1.5% or atleast 2.0%, on a dry weight basis. The potato tuber preferably has anincreased level of monounsaturated fatty acids (MUFA) and/or a lowerlevel of polyunsaturated fatty acids (PUFA) in both the total fatty acidcontent and in the TAG fraction of the total fatty acid content, such asan increased level of oleic acid and a reduced level of ALA, whencompared to a corresponding potato tuber lacking the geneticmodifications and/or exogenous polynucleotide(s). Preferably, the ALAlevel in the total fatty acid content of the tuber is reduced to lessthan 10% and/or the level of oleic acid in the total fatty acid contentis increased to at least 5%, preferably at least 10% or more preferablyat least 15%, when compared to a corresponding potato tuber lacking thegenetic modifications and/or exogenous polynucleotide(s). Furthermore,in an embodiment the level of palmitic acid in the total fatty acidcontent of the tuber is increased and/or the stearic acid (18:0) levelsdecreased in the total fatty acid content of the tuber, when compared toa corresponding potato tuber lacking the genetic modifications and/orexogenous polynucleotide(s). In an embodiment, the starch content of thetuber is between about 90% and 100% on a weight basis relative to awild-type tuber when they are grown under the same conditions.

In an embodiment, the potato plant, or part thereof preferably a tuber,of the invention comprises

a) a first exogenous polynucleotide which encodes a transcription factorpolypeptide that increases the expression of one or more glycolyticand/or fatty acid biosynthetic genes in the tuber, and

b) a second exogenous polynucleotide which encodes a polypeptideinvolved in the biosynthesis of one or more non-polar lipids,

wherein each exogenous polynucleotide is operably linked to a promoterwhich is capable of directing expression of the polynucleotide in thetuber during growth of the potato plant.

In a preferred embodiment, the potato tuber further comprises one ormore or all of

c) a third exogenous polynucleotide which encodes an oil body coating(OBC) polypeptide, preferably an LDAP,

d) a first genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in the catabolismof triacylglycerols (TAG) in the tuber when compared to a correspondingtuber lacking the first genetic modification, for example where thepolypeptide is SDP1,

e) a fourth exogenous polynucleotide which encodes a polypeptide whichincreases the export of fatty acids out of plastids of the tuber whencompared to a corresponding tuber lacking the fourth exogenouspolynucleotide,

f) a second genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in importing fattyacids into plastids of the tuber when compared to a corresponding tuberlacking the second genetic modification, and

g) a third genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in diacylglycerol(DAG) production in plastids of the tuber when compared to acorresponding tuber lacking the third genetic modification.

In further embodiments, additional genetic modifications in the tuberare as defined in the context of a cell or plant of the invention.

In a sixteenth aspect, the present invention provides a sorghum orsugarcane plant, or part thereof preferably a stem or a leaf, which hasa total fatty acid content of at least 6% or at least 8% on a dry weightbasis and/or a TAG content in the stem of at least 2% or at least 3% ona dry weight basis and/or has an increase in TAG content of at least50-fold in the stem and/or at least 100-fold in leaf on a weight basis.In embodiments, the sorghum or sugarcane plant, or part thereofpreferably a stem or a leaf, is characterised by features as defined inthe context of a cell or plant or part thereof of the invention.

In a seventeenth aspect, the present invention provides a sorghum orsugarcane plant, or part thereof preferably a stem or leaf, whichcomprises

a) a first exogenous polynucleotide which encodes a transcription factorpolypeptide that increases the expression of one or more glycolyticand/or fatty acid biosynthetic genes in the plant or part thereof, and

b) a second exogenous polynucleotide which encodes a polypeptideinvolved in the biosynthesis of one or more non-polar lipids, whereineach exogenous polynucleotide is operably linked to a promoter which iscapable of directing expression of the polynucleotide in the plant orpart thereof during growth of the plant.

Preferably, the promoter which directs expression of at least the firstexogenous polynucleotide is a promoter other than a rice ubiquitinpromoter (Rubi3). More preferably, the promoter is not a ubiquitinpromoter or any other constitutive promoter. Preferably, the first andsecond exogenous polynucleotides and their respective promoters arelinked on one genetic construct which is integrated into the plantgenome.

In an embodiment, the sugar content of the sugarcane stem is betweenabout 70% and 100% on a weight basis relative to a wild-type sugarcanestem when they are grown under the same conditions. Alternatively, thesugar content is between 50% and 70%.

In an embodiment, the sorghum or sugarcane plant, or part thereofpreferably a stem or leaf, of the invention comprises

a) a first exogenous polynucleotide which encodes a transcription factorpolypeptide that increases the expression of one or more glycolyticand/or fatty acid biosynthetic genes in the stem(s) of the plant, and

b) a second exogenous polynucleotide which encodes a polypeptideinvolved in the biosynthesis of one or more non-polar lipids,

wherein at least one of the exogenous polynucleotides, preferably atleast the first exogenous polynucleotide, is operably linked to apromoter which is preferentially expressed in the stem(s) relative tothe leaves during growth of the plant.

In an embodiment, the sorghum or sugarcane plant or part thereof of theinvention further comprises one or more or all of

c) a third exogenous polynucleotide which encodes an oil body coating(OBC) polypeptide, preferably an LDAP,

d) a first genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in the catabolismof triacylglycerols (TAG) in the plant or part thereof when compared toa corresponding plant or part thereof lacking the first geneticmodification,

e) a fourth exogenous polynucleotide which encodes a polypeptide whichincreases the export of fatty acids out of plastids of the plant or partthereof when compared to a corresponding plant or part thereof lackingthe fourth exogenous polynucleotide,

f) a second genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in importing fattyacids into plastids of the plant or part thereof when compared to acorresponding plant or part thereof lacking the second geneticmodification, and

g) a third genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in diacylglycerol(DAG) production in plastids of the plant or part thereof when comparedto a corresponding plant or part thereof lacking the third geneticmodification.

The sorghum or sugarcane plant or part thereof of the inventionpreferably has an increased level of monounsaturated fatty acids (MUFA)and/or a lower level of polyunsaturated fatty acids (PUFA) in both thetotal fatty acid content and in the TAG fraction of the total fatty acidcontent, such as an increased level of oleic acid and a reduced level ofALA, when compared to a corresponding plant or part thereof lacking thegenetic modifications and/or exogenous polynucleotide(s).

Preferably, the ALA level in the total fatty acid content is less than10% and/or the level of oleic acid in the total fatty acid content is atleast 5%, preferably at least 10% or more preferably at least 15%, whencompared to a corresponding plant or part thereof lacking the geneticmodifications and/or exogenous polynucleotide(s).

In further embodiments, additional genetic modifications in the sorghumor sugarcane plant or part thereof are as defined in the context of acell or plant of the invention.

In a eighteenth aspect, the present invention provides a transgenicmonocotyledonous plant, or part thereof preferably a leaf, a grain, astem, a root or an endosperm, which has a total fatty acid content orTAG content which is increased at least 5-fold on a weight basis whencompared to a corresponding non-transgenic monocotyledonous plant, orpart thereof. Alternatively, the invention provides a transgenicmonocotyledonous plant whose endosperm has a TAG content which is atleast 2.0%, preferably at least 3%, more preferably at least 4% or atleast 5%, on a weight basis, or part of the plant, preferably a leaf, astem, a root, a grain or an endosperm. In an embodiment, the endospermhas a TAG content of at least 2% which is increased at least 5-foldrelative to a corresponding non-transgenic endosperm. Preferably, theplant is fully male and female fertile, its pollen is essentially 100%viable, and its grain has a germination rate which is between 70% and100% relative to corresponding wild-type grain. In an embodiment, thetransgenic plant is a progeny plant at least two generations derivedfrom an initial transgenic wheat plant, and is preferably homozygous forthe transgenes. In embodiments, the monocotyledonous plant, or partthereof preferably a leaf, stem, grain or endosperm, is furthercharacterised by one or more features as defined in the context of acell or plant of the invention.

In an nineteenth aspect, the present invention provides amonocotyledonous plant, or part thereof preferably a leaf, a grain, stemor an endosperm, which comprises

a) a first exogenous polynucleotide which encodes a transcription factorpolypeptide that increases the expression of one or more glycolyticand/or fatty acid biosynthetic genes in the plant or part thereof, and

b) a second exogenous polynucleotide which encodes a polypeptideinvolved in the biosynthesis of one or more non-polar lipids,

wherein each exogenous polynucleotide is operably linked to a promoterwhich is capable of directing expression of the polynucleotide in theplant or part thereof during growth of the plant.

Preferably, the promoter which directs expression of at least the firstexogenous polynucleotide is a promoter other than a constitutivepromoter.

In an embodiment, the starch content of the grain of a monocotyledonousplant of the invention is between about 70% and 100% on a weight basisrelative to a wild-type grain when the plants from which they areobtained are grown under the same conditions. Preferred monocotyledonousplants in the above two aspects are wheat, rice, sorghum and corn(maize).

In an embodiment, the monocotyledonous plant, or part thereof,preferably a leaf, a grain or endosperm, of the invention comprises

a) a first exogenous polynucleotide which encodes a transcription factorpolypeptide that increases the expression of one or more glycolyticand/or fatty acid biosynthetic genes in the endosperm of the plant, and

b) a second exogenous polynucleotide which encodes a polypeptideinvolved in the biosynthesis of one or more non-polar lipids,

wherein at least one of the exogenous polynucleotides, preferably atleast the first exogenous polynucleotide, is operably linked to apromoter which is expressed at a greater level in the endosperm relativeto the leaves during growth of the plant.

In a preferred embodiment, the monocotyledonous plant or part thereoffurther comprises one or more or all of

c) a third exogenous polynucleotide which encodes an oil body coating(OBC) polypeptide, preferably an LDAP,

d) a first genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in the catabolismof triacylglycerols (TAG) in the plant or part thereof when compared toa corresponding plant or part thereof lacking the first geneticmodification,

e) a fourth exogenous polynucleotide which encodes a polypeptide whichincreases the export of fatty acids out of plastids of the plant or partthereof when compared to a corresponding plant or part thereof lackingthe fourth exogenous polynucleotide,

f) a second genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in importing fattyacids into plastids of the plant or part thereof when compared to acorresponding plant or part thereof lacking the second geneticmodification, and

g) a third genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in diacylglycerol(DAG) production in plastids of the plant or part thereof when comparedto a corresponding plant or part thereof lacking the third geneticmodification.

In an embodiment, the monocotyledonous plant comprises features a), b),one or both of d) and e), and optionally one of c), f) and g).

The monocotyledonous plant, or part thereof preferably a leaf, a grain,stem or an endosperm of the invention preferably has an increased levelof monounsaturated fatty acids (MUFA) and/or a lower level ofpolyunsaturated fatty acids (PUFA) in both the total fatty acid contentand in the TAG fraction of the total fatty acid content, such as forexample an increased level of oleic acid and a reduced level of LA(18:2), when compared to a corresponding plant or part thereof lackingthe genetic modifications and/or exogenous polynucleotide(s).

Preferably, the linoleic acid (LA, 18:2) level in the total fatty acidcontent of the grain or endosperm is reduced by at least 5% and/or thelevel of oleic acid in the total fatty acid content is increased by atleast 5% relative to a corresponding wild-type plant or part thereof,preferably at least 10% or more preferably at least 15%, when comparedto a corresponding plant or part thereof lacking the geneticmodifications and/or exogenous polynucleotide(s).

The following embodiments apply to each of the plants and parts thereofof the fifteenth, sixteenth, seventeenth, eighteenth and nineteenthaspects.

In an embodiment, the polypeptide involved in the biosynthesis of one ormore non-polar lipids is a DGAT or a PDAT and the OBC polypeptide is anoleosin. Alternatively, the OBC polypeptide is an LDAP.

In an embodiment, the transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the plant or part thereof is a WRI1 polypeptide and thepolypeptide involved in the biosynthesis of one or more non-polar lipidsis a DGAT or a PDAT.

In an embodiment, the transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the plant or part thereof is a WRI1 polypeptide, a LEC2polypeptide, a LEC1 polypeptide or a LEC1-like polypeptide and the OBCpolypeptide is an oleosin. Alternatively, the OBC polypeptide is anLDAP.

In an embodiment, the transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the plant or part thereof is a WRI1 polypeptide, a LEC2polypeptide, a LEC1 polypeptide or a LEC1-like polypeptide, thepolypeptide involved in the biosynthesis of one or more non-polar lipidsis a DGAT or a PDAT and the OBC polypeptide is an oleosin.Alternatively, the OBC polypeptide is an LDAP.

In an embodiment, the plant or part thereof comprises two exogenouspolynucleotides encoding two different transcription factor polypeptidesthat increase the expression of one or more glycolytic and/or fatty acidbiosynthetic genes in the cell, such as WRI1 and LEC2, or WRI1 and LEC1.

In each of the embodiments of the cells, plants and parts thereof of theinvention (including of the second, third, fourth, fifth, sixth,seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth,fourteenth, fifteenth sixteenth, seventeenth, eighteenth and nineteenthaspects), it is preferred that the transcription factor polypeptide thatincreases the expression of one or more glycolytic and/or fatty acidbiosynthetic genes in the cell is a WRI1 polypeptide, a LEC2polypeptide, a LEC1 polypeptide or a LEC1-like polypeptide, thepolypeptide involved in the biosynthesis of one or more non-polar lipidsis a DGAT or a PDAT and the polypeptide involved in the catabolism oftriacylglycerols (TAG) in the cell is an SDP1 lipase.

In each of the embodiments of the cells, plants and parts thereof of theinvention (including of the second, third, fifth, sixth, seventh,eighth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth,sixteenth, seventeenth, eighteenth and nineteenth aspects, but excludingthe fifth and tenth aspects), it is preferred that the transcriptionfactor polypeptide that increases the expression of one or moreglycolytic and/or fatty acid biosynthetic genes in the cell is a WRI1polypeptide, a LEC2 polypeptide, a LEC1 polypeptide or a LEC1-likepolypeptide, the polypeptide involved in the biosynthesis of one or morenon-polar lipids is a DGAT or a PDAT and the polypeptide which increasesthe export of fatty acids out of plastids of the cell is a fatty acidthioesterase, preferably a FATA or a FATB polypeptide, more preferably aFATA polypeptide or a fatty acid thioesterase other than a medium chainfatty acid thioesterase.

In each of the above embodiments of the cells, plants and parts thereofof the invention (including of the second, third, fourth, fifth, sixth,seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth,fourteenth, fifteenth, sixteenth seventeenth, eighteenth and nineteenthaspects), it is preferred that the transcription factor polypeptide thatincreases the expression of one or more glycolytic and/or fatty acidbiosynthetic genes in the plant or part thereof is a combination of atleast two polypeptides, preferably a WRI1 polypeptide and a LEC2polypeptide. More preferably, said at least two transcription factorpolypeptides are expressed from different promoters. Most preferably,the exogenous polynucleotides encoding said at least two polypeptidesare linked on a single genetic construct integrated into the cell orplant genome.

In each of the above embodiments, when the plant is a dicotyledonousplant, said transcription factor may be a monocotyledonous planttranscription factor. Conversely, when the plant is a monocotyledonousplant, said transcription factor may be a dicotyledonous planttranscription factor. Said transcription factor is preferably atranscription factor other than A. thaliana WRI1 (SEQ ID NOs: 21 or 22).

In each of the above embodiments, it is preferred that the plant is atransgenic progeny plant at least two generations derived from aninitial transgenic plant, and is preferably homozygous for thetransgenes.

In further embodiments, additional genetic modifications in the plant orpart thereof are as defined in the context of a cell of the invention.

In an embodiment, a plant, or part thereof, of the invention (includingof the sixth, seventh, eighth, ninth, tenth, eleventh, twelfth,thirteenth, fourteenth, fifteenth, sixteenth seventeenth, eighteenth andnineteenth aspects) has one or more or all of the following features(where applicable);

i) the plant comprises a part, preferably a vegetative part, which hasan increased synthesis of total fatty acids relative to a correspondingpart lacking the first exogenous polynucleotide, or a decreasedcatabolism of total fatty acids relative to a corresponding part lackingthe first exogenous polynucleotide, or both, such that it has anincreased level of total fatty acids relative to a corresponding partlacking the first exogenous polynucleotide,

ii) the plant comprises a part, preferably a vegetative part, which hasan increased expression and/or activity of a fatty acyl acyltransferasewhich catalyses the synthesis of TAG, DAG or MAG, preferably TAG,relative to a corresponding part having the first exogenouspolynucleotide and lacking the exogenous polynucleotide which encodes apolypeptide involved in the biosynthesis of one or more non-polarlipids,

iii) the plant comprises a part, preferably a vegetative part, which hasa decreased production of lysophosphatidic acid (LPA) from acyl-ACP andG3P in its plastids relative to a corresponding part having the firstexogenous polynucleotide and lacking the genetic modification whichdown-regulates endogenous production and/or activity of a polypeptideinvolved in diacylglycerol (DAG) production in plastids in the plantpart,

iv) the plant comprises a part, preferably a vegetative part, which hasan altered ratio of C16:3 to C18:3 fatty acids in its total fatty acidcontent and/or its galactolipid content relative to a corresponding partlacking the exogenous polynucleotide(s) and/or genetic modification(s),preferably a decreased ratio,

v) a vegetative part of the plant comprises a total non-polar lipidcontent of at least about 8%, at least about 10%, at least about 11%, atleast about 12%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, between 8% and 75%, between10% and 75%, between 11% and 75%, between about 15% and 75%, betweenabout 20% and 75%, between about 30% and 75%, between about 40% and 75%,between about 50% and 75%, between about 60% and 75%, or between about25% and 50% (w/w dry weight), preferably before flowering,

vi) a vegetative part of the plant comprises a TAG content of at leastabout 8%, at least about 10%, at least about 11%, at least about 12%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, between 8% and 75%, between 10% and 75%,between 11% and 75%, between about 15% and 75%, between about 20% and75%, between about 30% and 75%, between about 40% and 75%, between about50% and 75%, between about 60% and 75%, or between about 25% and 50%(w/w dry weight), preferably before flowering,

vii) the transcription factor polypeptide(s) is selected from the groupconsisting of WRI1, LEC1, LEC1-like, LEC2, BBM, FUS3, ABI3, ABI4, ABI5,Dof4 and Dof11, preferably WRI1, LEC1 or LEC2, or the group consistingof MYB73, bZIP53, AGL15, MYB115, MYB118, TANMEI, WUS, GFR2a1, GFR2a2 andPHR1,

viii) oleic acid comprises at least 20% (mol %), at least 22% (mol %),at least 30% (mol %), at least 40% (mol %), at least 50% (mol %), or atleast 60% (mol %), preferably about 65% (mol %) or between 20% and about65% of the total fatty acid content in the plant, or part thereof,

ix) non-polar lipid in the plant, or part thereof preferably avegetative part, comprises an increased level of one or more fatty acidswhich comprise a hydroxyl group, an epoxy group, a cyclopropane group, adouble carbon-carbon bond, a triple carbon-carbon bond, conjugateddouble bonds, a branched chain such as a methylated or hydroxylatedbranched chain, or a combination of two or more thereof. or any of two,three, four, five or six of the aforementioned groups, bonds or branchedchains,

x) non-polar lipid in the plant, or part thereof preferably a vegetativepart, comprises one or more polyunsaturated fatty acids selected fromeicosadienoic acid (EDA), arachidonic acid (ARA), stearidonic acid(SDA), eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA),eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA),docosahexaenoic acid (DHA), or a combination of two of more thereof,

xi) the part is a vegetative plant part, such as a leaf or a stem, orpart thereof,

xii) one or more or all of the promoters are selected from promoterother than a constitutive promoter, preferably a tissue-specificpromoter such as a leaf and/or stem specific promoter, a developmentallyregulated promoter such as a senescense-specific promoter such as aSAG12 promoter, an inducible promoter, or a circadian-rhythm regulatedpromoter, preferably wherein at least one of the promoters operablylinked to an exogenous polynucleotide which encodes a transcriptionfactor polypeptide is a promoter other than a constitutive promoter,

xiii) the plant, or part thereof preferably a vegetative part, comprisesa total fatty acid content which comprises medium chain fatty acids,preferably C12:0, C14:0 or both, at a level of at least 5% of the totalfatty acid content and optionally an exogenous polynucleotide whichencodes an LPAAT which has preferential activity for fatty acids with amedium chain length (C8 to C14), preferably C12:0 or C14:0,

xiv) the plant, or part thereof preferably a vegetative part, comprisesa total fatty acid content whose oleic acid level and/or palmitic acidlevel is increased by at least 2% relative to a corresponding plant, orpart thereof, lacking the exogenous polynucleotide(s) and/or geneticmodification(s), and/or whose α-linolenic acid (ALA) level and/orlinoleic acid level is decreased by at least 2% relative to acorresponding plant, or part thereof, lacking the exogenouspolynucleotide(s) and/or genetic modification(s),

xv) non-polar lipid in the plant, or part thereof preferably avegetative part, comprises a modified level of total sterols, preferablyfree (non-esterified) sterols, steroyl esters, steroyl glycosides,relative to the non-polar lipid in a corresponding plant, or partthereof, lacking the exogenous polynucleotide(s) and/or geneticmodification(s),

xvi) non-polar lipid in the plant, or part thereof, comprises waxesand/or wax esters,

xvii) the plant, or part thereof preferably a vegetative part, is onemember of a population or collection of at least about 1000 such plants,or parts thereof,

xviii) the plant comprises an exogenous polynucleotide encoding asilencing suppressor, wherein the exogenous polynucleotide is operablylinked to a promoter which is capable of directing expression of thepolynucleotide in the plant,

xix) the level of one or more non-polar lipid(s) and/or the totalnon-polar lipid content of the plant or part thereof, preferably avegetative plant part, is at least 2% greater on a weight basis than ina corresponding plant or part, respectively, which comprises exogenouspolynucleotides encoding an Arabidposis thaliana WRI1 (SEQ ID NO:21) andan Arabidopsis thaliana DGAT1 (SEQ ID NO:1),

xx) a total polyunsaturated fatty acid (PUFA) content which is decreasedrelative to the total PUFA content of a corresponding plant lacking theexogenous polynucleotide(s) and/or genetic modification(s),

xxi) the plant part is a potato (Solanum tuberosum) tuber, a sugarbeet(Beta vulgaris) beet, a sugarcane (Saccharum sp.) or sorghum (Sorghumbicolor) stem, a monocotyledonous plant seed having an increased totalfatty acid content in its endosperm such as, for example, a wheat(Triticum aestivum) grain or a corn (Zea mays) kernel, a Nicotiana spp.leaf, or a legume seed having an increased total fatty acid content suchas, for example, a Brassica sp. seed or a soybean (Glycine max) seed,

xxii) if the plant part is a seed, the seed germinates at a ratesubstantially the same as for a corresponding wild-type seed or whensown in soil produces a plant whose seed germinate at a ratesubstantially the same as for corresponding wild-type seed, and

xxiii) the plant is an algal plant such as from diatoms(bacillariophytes), green algae (chlorophytes), blue-green algae(cyanophytes), golden-brown algae (chrysophytes), haptophytes, brownalgae or heterokont algae.

In the above embodiments, a preferred plant part is a leaf piece havinga surface area of at least 1 cm² or a stem piece having a length of atleast 1 cm.

In an embodiment of the above aspects, the plant or plant part has beentreated so it is no longer able to be propagated or give rise to aliving plant, i.e. it is dead. For example, the plant or plant part hasbeen dried and/or ground.

In the above embodiments, it is preferred that the total non-polar lipidcontent of the plant part is at least 3% greater, more preferably atleast 5% greater, than the total non-polar lipid content in acorresponding plant part transformed with genes encoding a WRI1 and aDGAT but lacking the other exogenous polynucleotides and geneticmodifications as described herein for the above aspects. Morepreferably, that degree of increase is in a stem or root of the plant.

In an embodiment, the addition of one or more of the exogenouspolynucleotides or genetic modifications, preferably the exogenouspolynucleotide encoding an OBC or a fatty acyl thioesterase or thegenetic modification which down-regulates endogenous production and/oractivity of a polypeptide involved in the catabolism of triacylglycerols(TAG) in the plant, more preferably the exogenous polynucleotide whichencodes a FATA thioesterase or an LDAP or which decreases expression ofan endogenous TAG lipase such as a SDP1 TAG lipase in the plant, resultsin a synergistic increase in the total non-polar lipid content of theplant part when added to the pair of transgenes WRI1 and DGAT,particularly before the plant flowers and even more particularly in thestems and/or roots of the plant. For example, see Examples 8, 11 and 15.In a preferred embodiment, the increase in the TAG content of the stemor root of the plant is at least 2-fold, more preferably at least3-fold, relative to a corresponding part transformed with genes encodingWRI1 and DGAT1 but lacking the FATA thioesterase, LDAP and the geneticmodification which down-regulates endogenous production and/or activityof a polypeptide involved in the catabolism of triacylglycerols (TAG) inthe plant. Most preferably, at least the promoter that directsexpression of the first exogenous polynucleotide is a promoter otherthan a constitutive promoter.

In the embodiments of the sixth, seventh, eighth, ninth, tenth,eleventh, twelfth, thirteenth, fourteenth, sixteenth, eighteenth andnineteenth aspects, it is preferred that the plant or the part thereofis phenotypically normal, in that it is not significantly reduced in itsability to grow and reproduce when compared to an unmodified plant orpart thereof. Preferably, the biomass, growth rate, germination rate,storage organ size, seed size and/or the number of viable seeds producedis not less than 90% of that of a corresponding wild-type plant whengrown under identical conditions. In an embodiment, the plant is maleand female fertile to the same extent as a corresponding wild-type plantand its pollen (if produced) is as viable as the pollen of thecorresponding wild-type plant, preferably about 100% viable. In anembodiment, the plant produces seed which has a germination rate of atleast 90% relative to the germination rate of corresponding seed of awild-type plant, where the plant species produces seed. In anembodiment, the plant of the invention has a plant height which is atleast 90% relative to the height of the corresponding wild-type plantgrown under the same conditions. A combination of each of these featuresis envisaged. In an alternative embodiment, the plant of the inventionhas a plant height which is between 60% and 90% relative to the heightof the corresponding wild-type plant grown under the same conditions. Inan embodiment, the plant or part thereof of the invention, preferably aplant leaf, does not exhibit increased necrosis, i.e. the extent ofnecrosis, if present, is the same as that exhibited by a correspondingwild-type plant or part thereof grown under the same conditions and atthe same stage of plant development. This feature applies in particularto the plant or part thereof comprising an exogenous polynucleotidewhich encodes a fatty acid thioesterase such as a FATB thioesterase.

The following embodiments apply to the plant, or part thereof, of theinvention (including of the sixth, seventh, eighth, ninth, tenth,eleventh, twelfth, thirteenth, fourteenth, sixteenth, eighteenth andnineteenth aspects), as well as a method of producing the plant or partthereof or a method of using same. In an embodiment, the polypeptideinvolved in the biosynthesis of one or more non-polar lipids is a fattyacyl acyltransferase involved in the biosynthesis of TAG, DAG ormonoacylglycerol (MAG) in the plant or part thereof, preferably of TAGin the plant or part thereof, such as a DGAT, PDAT, LPAAT, GPAT or MGAT,preferably a DGAT or a PDAT.

In another embodiment, the polypeptide involved in the catabolism oftriacylglycerols (TAG) in the plant or plant part is an SDP1 lipase, aCgi58 polypeptide, an acyl-CoA oxidase such as ACX1 or ACX2, or apolypeptide involved in β-oxidation of fatty acids in the plant such asa PXA1 peroxisomal ATP-binding cassette transporter, preferably an SDP1lipase.

In an embodiment, the oil body coating (OBC) polypeptide is oleosin,such as a polyoleosin or a caleosin, or preferably a lipid dropletassociated protein (LDAP).

In an embodiment, the polypeptide which increases the export of fattyacids out of plastids of the plant is a C16 or C18 fatty acidthioesterase such as a FATA polypeptide or a FATB polypeptide, a fattyacid transporter such as an ABCA9 polypeptide or a long-chain acyl-CoAsynthetase (LACS).

In an embodiment, the polypeptide involved in importing fatty acids intoplastids of the plant is a fatty acid transporter, or subunit thereof,preferably a TGD polypeptide such as, for example, a TGD1 polypeptide, aTGD2 polypeptide, a TGD3 polypeptide or a TGD4 polypeptide.

In an embodiment, the polypeptide involved in diacylglycerol (DAG)production in the plastid is a plastidial GPAT, a plastidial LPAAT or aplastidial PAP.

In an embodiment, the plant, or part thereof, of the invention is a 16:3plant, or part thereof, and which comprises one or more or all of thefollowing;

a) an exogenous polynucleotide which encodes a polypeptide whichincreases the export of fatty acids out of plastids of the plant whencompared to a corresponding plant lacking the exogenous polynucleotide,

b) a first genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in importing fattyacids into plastids of the plant when compared to a corresponding plantlacking the first genetic modification, and

c) a second genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in diacylglycerol(DAG) production in the plastid when compared to a corresponding plantlacking the second genetic modification,

wherein the exogenous polynucleotide is operably linked to a promoterwhich is capable of directing expression of the polynucleotide in theplant, or part thereof.

In an alternative embodiment, the plant, or part thereof, of theinvention is a 18:3 plant or part thereof.

In an embodiment, before the plant flowers, a vegetative part of theplant comprises a total non-polar lipid content of at least about 8%, atleast about 10%, about 11%, between 8% and 15%, or between 9% and 12%(w/w dry weight).

In an embodiment, one or more or all of the genetic modifications is amutation of an endogenous gene which partially or completely inactivatesthe gene, such as a point mutation, an insertion, or a deletion (or acombination of one or more thereof), preferably an introduced mutation.The point mutation may be a premature stop codon, a splice-sitemutation, a frame-shift mutation or an amino acid substitution mutationthat reduces activity of the gene or the encoded polypeptide. Thedeletion may be of one or more nucleotides within a transcribed exon orpromoter of the gene, or extend across or into more than one exon, orextend to deletion of the entire gene. Preferably the deletion isintroduced by use of ZF, TALEN or CRISPR technologies. In an alternateembodiment, one or more or all of the genetic modifications is anexogenous polynucleotide encoding an RNA molecule which inhibitsexpression of the endogenous gene, wherein the exogenous polynucleotideis operably linked to a promoter which is capable of directingexpression of the polynucleotide in the plant, or part thereof.

In an embodiment, the exogenous polynucleotide encoding WRI1 comprisesone or more of the following:

i) nucleotides encoding a polypeptide comprising amino acids whosesequence is set forth as any one of SEQ ID NOs:21 to 75 or 205 to 210,or a biologically active fragment thereof, or a polypeptide whose aminoacid sequence is at least 30% identical to any one or more of SEQ IDNOs: 21 to 75 or 205 to 210,

ii) nucleotides whose sequence is at least 30% identical to i), and

iii) nucleotides which hybridize to i) and/or ii) under stringentconditions.

Preferably, the WRI1 polypeptide is a WRI1 polypeptide other thanArabidopsis thaliana WRI1 (SEQ ID NOs:21 or 22). More preferably, theWRI1 polypeptide comprises amino acids whose sequence is set forth asSEQ ID NO:208, or a biologically active fragment thereof, or apolypeptide whose amino acid sequence is at least 30% identical thereto.

In an embodiment, the total non-polar lipid content, or the one or morenon-polar lipids, and/or the level of the oleic acid or a PUFA in theplant or part thereof is determinable by analysis by using gaschromatography of fatty acid methyl esters obtained from the plant orvegetative part thereof.

In a further embodiment, wherein the plant part is a leaf and the totalnon-polar lipid content of the leaf is determinable by analysis usingNuclear Magnetic Resonance (NMR).

In an embodiment, the plant, or part thereof, is a member of apopulation or collection of at least about 1000 such plants or parts.

In a further aspect, the present invention provides a population of atleast about 1000 plants, each being a plant of the invention, growing ina field.

In another aspect, the present invention provides a collection of atleast about 1000 vegetative plant parts, each being a vegetative plantpart of the invention, wherein the vegetative plant parts have beenharvested from plants growing in a field.

In an embodiment of the cell, non-human organism, plant or part thereofof the invention, the transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the cell is a WRI1 transcription factor, the polypeptideinvolved in the biosynthesis of one or more non-polar lipids is a DGATsuch as a DGAT1 or a DGAT2, or a PDAT, and the polypeptide involved inthe catabolism of triacylglycerols (TAG) in the cell is an SDP1 lipase.In a preferred embodiment, the oil body coating (OBC) polypeptide is anoleosin, the polypeptide which increases the export of fatty acids outof plastids of the cell is a fatty acid thioesterase such as a FATA orFATB thioesterase, the polypeptide involved in importing fatty acidsinto plastids of the cell is a TGD polypeptide, preferably a TGD1polypeptide, and the polypeptide involved in diacylglycerol (DAG)production in the plastid is a plastidial GPAT. In a more preferredembodiment, the cell is in a vegetative plant part and the TAG contentof the vegetative plant part prior to flowering of the plant is at least8% (% dry weight).

In an embodiment, the plant, vegetative plant part, non-human organismor part thereof, seed or potato tuber comprises a first exogenouspolynucleotide encoding a WRI1, a second exogenous polynucleotideencoding a DGAT or a PDAT, preferably a DGAT1, a third exogenouspolynucleotide encoding an RNA which reduces expression of a geneencoding an SDP1 polypeptide, and a fourth exogenous polynucleotideencoding an oleosin. In preferred embodiments, the vegetative plantpart, non-human organism or part thereof, seed or potato tuber has oneor more or all of the following features:

i) a total lipid content of at least 8%, at least 10%, at least 12%, atleast 14%, or at least 15.5% (% weight),

ii) at least a 3 fold, at least a 5 fold, at least a 7 fold, at least an8 fold, or least a 10 fold, at higher total lipid content in thevegetative plant part or non-human organism relative to a correspondingvegetative plant part or non-human organism lacking the exogenouspolynucleotides,

iii) a total TAG content of at least 5%, at least 6%, at least 6.5% orat least 7% (% weight of dry weight or seed weight),

iv) at least a 40 fold, at least a 50 fold, at least a 60 fold, or atleast 70 fold, at least 100 fold, or at least a 120-fold higher totalTAG content relative to a corresponding vegetative plant part ornon-human organism lacking the exogenous polynucleotides,

v) oleic acid comprises at least 15%, at least 19% or at least 22% (%weight of dry weight or seed weight) of the fatty acids in TAG,

vi) at least a 10 fold, at least a 15 fold or at least a 17 fold higherlevel of oleic acid in TAG relative to a corresponding vegetative plantpart or non-human organism lacking the exogenous polynucleotides,

vii) palmitic acid comprises at least 20%, at least 25%, at least 30% orat least 33% (% weight) of the fatty acids in TAG,

viii) at least a 1.5 fold higher level of palmitic acid in TAG relativeto a corresponding vegetative plant part or non-human organism lackingthe exogenous polynucleotides,

ix) linoleic acid comprises at least 22%, at least 25%, at least 30% orat least 34% (% weight) of the fatty acids in TAG,

x) α-linolenic acid comprises less than 20%, less than 15%, less than11% or less than 8% (% weight) of the fatty acids in TAG,

xi) at least a 5 fold, or at least an 8 fold, lower level of α-linolenicacid in TAG relative to a corresponding vegetative plant part ornon-human organism lacking the exogenous polynucleotides, and

xii) for a potato tuber, a TAG content of at least 0.5% on a dry weightbasis and/or a total fatty acid content of at least 1%, preferably atleast 1.5% or at least 2.0%, on a dry weight basis.

Also provided is seed of, or obtained from, a plant of the invention.

In another aspect, the invention provides a transgenic plant stem, orpart of a stem of at least 1 g dry weight, whose TAG content is at least5% on a weight basis (dry weight), preferably at least 6%, morepreferably at least 7%. In an embodiment, the transgenic plant stem orstem part is of, or preferably harvested from, a dicotyledonous plant.Alternatively, the transgenic, plant stem or stem part is of, orpreferably harvested from, a monocotyledonous plant. In an embodiment,the plant stem or stem part is of or from a plant other than sugarcane.In embodiments, the plant stem or stem part is further characterised byone or more features as defined in the context of a cell or plant of theinvention.

In another aspect, the invention provides a plant cell comprising

a) a first exogenous polynucleotide which encodes a PDAT,

b) a first genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in importing fattyacids into plastids of the cell, preferably a TGD polypeptide, whencompared to a corresponding cell lacking the first genetic modification,and one or more of

c) a second genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in the catabolismof triacylglycerols (TAG) in the cell, preferably an SDP1 polypeptide,when compared to a corresponding cell lacking the genetic modification,

d) a second exogenous polynucleotide which encodes a polypeptide whichincreases the export of fatty acids out of plastids of the cell,preferably a fatty acyl thioesterase, when compared to a correspondingcell lacking the second exogenous polynucleotide, and

e) a third genetic modification which down-regulates endogenousproduction and/or activity of a polypeptide involved in diacylglycerol(DAG) production in the plastid when compared to a corresponding celllacking the third genetic modification, wherein each exogenouspolynucleotide is operably linked to a promoter which is capable ofdirecting expression of the polynucleotide in the cell. In a preferredembodiment, the presence in the cell of the first, second or thirdgenetic modification or the second exogenous polynucleotidesynergistically increases the total non-polar lipid content of the cellwhen compared to a corresponding cell having the PDAT but lacking theadditional genetic modification or exogenous polynucleotide. Morepreferably, at least one of the exogenous polynucleotides is expressedfrom a promoter other than a constitutive promoter.

In another aspect, the present invention provides a process forobtaining a recombinant eukaryotic cell of the invention, the processcomprising the steps of:

i) introducing into a eukaryotic cell at least one exogenouspolynucleotide and/or at least one genetic modification as definedherein to produce a eukaryotic cell comprising a set of exogenouspolynucleotides and/or genetic modifications as defined herein,

ii) expressing the exogenous polynucleotide(s) in the cell or a progenycell thereof,

iii) analysing the lipid content of the cell or progeny cell, and

iv) selecting a cell of the invention.

In an embodiment, the one or more exogenous polynucleotides are stablyintegrated into the genome of the cell or progeny cell.

In an embodiment, the process further comprises the step of regeneratinga transgenic plant from the cell or progeny cell comprising the one ormore exogenous polynucleotides.

In a further embodiment, the step of regenerating a transgenic plant isperformed prior to the step of expressing the one or more exogenouspolynucleotides in the cell or a progeny cell thereof, and/or prior tothe step of analysing the lipid content of the cell or progeny cell,and/or prior to the step of selecting the cell or progeny cell having anincreased level of one or more non-polar lipids.

In another embodiment, the process further comprises a step of obtainingseed or a progeny plant from the transgenic plant, wherein the seed orprogeny plant comprises the one or more exogenous polynucleotides.

In yet another embodiment, the selected cell or regenerated planttherefrom, or a vegetative plant part or seed of the regenerated plant,has one or more of the features as defined herein.

In a further aspect, the present invention provides a method ofproducing a plant which has integrated into its genome a set ofexogenous polynucleotides and/or genetic modifications as definedherein, the method comprising the steps of

i) crossing two parental plants, wherein one plant comprises at leastone of the exogenous polynucleotides and/or at least one geneticmodifications as defined herein, and the other plant comprises at leastone of the exogenous polynucleotides and/or at least one geneticmodifications as defined herein, and wherein between them the twoparental plants comprise a set of exogenous polynucleotides and/orgenetic modifications as defined herein,

ii) screening one or more progeny plants from the cross for the presenceor absence of the set of exogenous polynucleotides and/or geneticmodifications as defined herein, and

iii) selecting a progeny plant which comprise the set of exogenouspolynucleotides and/or genetic modifications as defined herein, therebyproducing the plant.

Also provided is a transgenic cell or transgenic plant obtained usingthe process of the invention, or a part thereof, obtained therefromwhich comprises the set of exogenous polynucleotides and/or geneticmodifications as defined herein.

Also provided is the use of a set of exogenous polynucleotides and/orgenetic modifications as defined herein for producing a transgenic cell,a transgenic non-human organism or a part thereof or a seed having anenhanced ability to produce one or more non-polar lipids relative to acorresponding cell, non-human organism or part thereof or seed lackingthe set of exogenous polynucleotides and/or genetic modifications,wherein each exogenous polynucleotide is operably linked to a promoterthat is capable of directing expression of the exogenous polynucleotidein the transgenic cell, transgenic non-human organism or a part thereofor seed.

Preferably, at least one of the promoters operably linked to anexogenous polynucleotide which encodes a transcription factorpolypeptide is a promoter other than a constitutive promoter.

In an embodiment, the transgenic cell, non-human organism or partthereof, or seed comprises one or more of the features defined herein.

In a further aspect, the present invention provides a process forproducing an industrial product, the process comprising the steps of:

i) obtaining a recombinant eukaryotic cell of the invention, atransgenic non-human organism or a part thereof of the invention, atransgenic plant or part thereof of the invention, a seed of theinvention, or a transgenic cell or transgenic plant or part thereof ofthe invention, and

ii) converting at least some of the lipid in the cell, non-humanorganism or part thereof, plant or part thereof, or seed, to theindustrial product by applying heat, chemical, or enzymatic means, orany combination thereof, to the lipid in situ in the non-human organismor part thereof, and

iii) recovering the industrial product, thereby producing the industrialproduct.

In a further aspect, the present invention provides a process forproducing an industrial product, the process comprising the steps of:

i) obtaining a recombinant eukaryotic cell of the invention, atransgenic non-human organism or a part thereof of the invention, atransgenic plant or part thereof of the invention, a seed of theinvention, or a transgenic cell or transgenic plant or part thereof ofthe invention, and

ii) physically processing the cell, non-human organism or part thereof,plant or part thereof or seed of step i),

iii) simultaneously or subsequently converting at least some of thelipid in the processed cell, non-human organism or part thereof, plantor part thereof, or seed, to the industrial product by applying heat,chemical, or enzymatic means, or any combination thereof, to the lipidin the processed cell, non-human organism or part thereof, plant or partthereof, or seed, and

iv) recovering the industrial product, thereby producing the industrialproduct.

In an embodiment, of the two above aspects, the plant part is avegetative plant part.

In a further aspect, the present invention provides a process forproducing an industrial product, the process comprising the steps of:

i) obtaining a vegetative plant part having a total non-polar lipidcontent of at least about 18%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, between 18% and 75%, betweenabout 20% and 75%, between about 30% and 75%, between about 40% and 75%,between about 50% and 75%, between about 60% and 75%, or between about25% and 50% (w/w dry weight),

ii) converting at least some of the lipid in the vegetative plant partto the industrial product by applying heat, chemical, or enzymaticmeans, or any combination thereof, to the lipid in situ in thevegetative plant part, and

iii) recovering the industrial product, thereby producing the industrialproduct.

In another aspect, the present invention provides a process forproducing an industrial product, the process comprising the steps of:

i) obtaining a vegetative plant part having a total non-polar lipidcontent of at least about 18%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, between 18% and 75%, betweenabout 20% and 75%, between about 30% and 75%, between about 40% and 75%,between about 50% and 75%, between about 60% and 75%, or between about25% and 50% (w/w dry weight),

ii) physically processing the vegetative plant part of step i),

iii) simultaneously or subsequently converting at least some of thelipid in the processed vegetative plant part to the industrial productby applying heat, chemical, or enzymatic means, or any combinationthereof, to the lipid in the processed vegetative plant part, and

iv) recovering the industrial product,

thereby producing the industrial product.

In yet a further aspect, the present invention provides a process forproducing an industrial product, the process comprising the steps of:

i) obtaining a vegetative plant part having a total non-polar lipidcontent of at least about 11%, at least about 12%, at least about 15%,at least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, between 8% and 75%, between 10% and 75%, between 11% and 75%,between about 15% and 75%, between about 20% and 75%, between about 30%and 75%, between about 40% and 75%, between about 50% and 75%, betweenabout 60% and 75%, or between about 25% and 50% (w/w dry weight),wherein the plant is a 16:3 plant or vegetative part thereof,

ii) converting at least some of the lipid in the vegetative plant partto the industrial product by applying heat, chemical, or enzymaticmeans, or any combination thereof, to the lipid in situ in thevegetative plant part, and

iii) recovering the industrial product, thereby producing the industrialproduct.

In another aspect, the present invention provides a process forproducing an industrial product, the process comprising the steps of:

i) obtaining a vegetative plant part having a total non-polar lipidcontent of at least about 11%, at least about 12%, at least about 15%,at least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, between 8% and 75%, between 10% and 75%, between 11% and 75%,between about 15% and 75%, between about 20% and 75%, between about 30%and 75%, between about 40% and 75%, between about 50% and 75%, betweenabout 60% and 75%, or between about 25% and 50% (w/w dry weight),wherein the plant is a 16:3 plant or vegetative part thereof,

ii) physically processing the vegetative plant part of step i),

iii) simultaneously or subsequently converting at least some of thelipid in the processed vegetative plant part to the industrial productby applying heat, chemical, or enzymatic means, or any combinationthereof, to the lipid in the processed vegetative plant part, and

iv) recovering the industrial product,

thereby producing the industrial product.

In an embodiment, the step of physically processing the cell, non-humanorganism or part thereof, plant or part thereof, or seed comprises oneor more of rolling, pressing, crushing or grinding the cell, non-humanorganism or part thereof, plant or part thereof, or seed.

In an embodiment, the process comprises the steps of:

(a) extracting at least some of the non-polar lipid content of the cell,non-human organism or part thereof, plant or part thereof, or seed asnon-polar lipid, and

(b) recovering the extracted non-polar lipid,

wherein steps (a) and (b) are performed prior to the step of convertingat least some of the lipid in the cell, non-human organism or partthereof, plant or part thereof, or seed to the industrial product.

In an embodiment, the extracted non-polar lipid comprisestriacylglycerols, wherein the triacylglycerols comprise at least 90%,preferably at least 95%, of the extracted lipid.

In an embodiment, the industrial product is a hydrocarbon product suchas fatty acid esters, preferably fatty acid methyl esters and/or a fattyacid ethyl esters, an alkane such as methane, ethane or a longer-chainalkane, a mixture of longer chain alkanes, an alkene, a biofuel, carbonmonoxide and/or hydrogen gas, a bioalcohol such as ethanol, propanol, orbutanol, biochar, or a combination of carbon monoxide, hydrogen andbiochar. In a preferred embodiment, the total fatty acid content of thevegetative plant part comprises at least 5% C12:0, C14:0 or the sum ofC12:0 and C14:0 is at least 5% of the total fatty acid content and theindustrial product produced from the lipid in the vegetative plant partis a component in an aviation fuel.

In a further aspect, the present invention provides a process forproducing extracted lipid, the process comprising the steps of:

i) obtaining a recombinant eukaryotic cell of the invention, atransgenic non-human organism or a part thereof of the invention, atransgenic plant or part thereof of the invention, a seed of theinvention, or a transgenic cell or transgenic plant or part thereof ofthe invention,

ii) extracting lipid from the cell, non-human organism or part thereof,plant or part thereof or seed, and

iii) recovering the extracted lipid, thereby producing the extractedlipid.

In a further aspect, the present invention provides a process forproducing extracted lipid, the process comprising the steps of:

i) obtaining a vegetative plant part having a total non-polar lipidcontent of at least about 18%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, between 18% and 75%, betweenabout 20% and 75%, between about 30% and 75%, between about 40% and 75%,between about 50% and 75%, between about 60% and 75%, or between about25% and 50% (w/w dry weight),

ii) extracting lipid from the vegetative plant part, and

iii) recovering the extracted lipid,

thereby producing the extracted lipid.

In a further aspect, the present invention provides a process forproducing extracted lipid, the process comprising the steps of:

i) obtaining a vegetative plant part having a total non-polar lipidcontent of at least about 11%, at least about 12%, at least about 15%,at least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, between 8% and 75%, between 10% and 75%, between 11% and 75%,between about 15% and 75%, between about 20% and 75%, between about 30%and 75%, between about 40% and 75%, between about 50% and 75%, betweenabout 60% and 75%, or between about 25% and 50% (w/w dry weight),wherein the plant is a 16:3 plant or vegetative part thereof,

ii) extracting lipid from the vegetative plant part, and

iii) recovering the extracted lipid,

thereby producing the extracted lipid.

In an embodiment, a process of extraction of the comprises one or moreof drying, rolling, pressing, crushing or grinding the cell, non-humanorganism or part thereof, plant or part thereof, or seed, and/orpurifying the extracted lipid or seedoil.

In an embodiment, the process uses an organic solvent in the extractionprocess to extract the oil.

In a further embodiment, the process comprises recovering the extractedlipid or oil by collecting it in a container and/or one or more ofdegumming, deodorising, decolourising, drying, fractionating theextracted lipid or oil, removing at least some waxes and/or wax estersfrom the extracted lipid or oil, or analysing the fatty acid compositionof the extracted lipid or oil.

In an embodiment, the volume of the extracted lipid or oil is at least 1litre.

In a further embodiment, one or more or all of the following featuresapply:

(i) the extracted lipid or oil comprises triacylglycerols, wherein thetriacylglycerols comprise at least 90%, preferably at least 95% or atleast 96%, of the extracted lipid or oil,

(ii) the extracted lipid or oil comprises free sterols, steroyl esters,steroyl glycosides, waxes or wax esters, or any combination thereof, and

(iii) the total sterol content and/or composition in the extracted lipidor oil is significantly different to the sterol content and/orcomposition in the extracted lipid or oil produced from a correspondingcell, non-human organism or part thereof, plant or part thereof, orseed.

In an embodiment, the process further comprises converting the extractedlipid or oil to an industrial product.

In an embodiment, the industrial product is a hydrocarbon product suchas fatty acid esters, preferably fatty acid methyl esters and/or a fattyacid ethyl esters, an alkane such as methane, ethane or a longer-chainalkane, a mixture of longer chain alkanes, an alkene, a biofuel, carbonmonoxide and/or hydrogen gas, a bioalcohol such as ethanol, propanol, orbutanol, biochar, or a combination of carbon monoxide, hydrogen andbiochar. In a preferred embodiment, the total fatty acid content of thevegetative plant part comprises at least 5% C12:0, C14:0 or the sum ofC12:0 and C14:0 is at least 5% of the total fatty acid content and theindustrial product produced from the lipid in the vegetative plant partis a component in an aviation fuel.

In a further embodiment, the plant part is an aerial plant part or agreen plant part, preferably a vegetative plant part such as a plantleaf or stem. In an alternative embodiment, the plant part is a tuber orbeet, such as a potato (Solanum tuberosum) tuber or a sugar beet.

In yet a further embodiment, the process further comprises a step ofharvesting the cell, non-human organism or part thereof, plant or partthereof such as a tuber or beet, or seed, preferably with a mechanicalharvester, or by a process comprising filtration, centrifugation,sedimentation, flotation or flocculation of algal or fungal organisms.

In another embodiment, the level of a lipid in the cell, non-humanorganism or part thereof, plant or part thereof, or seed and/or in theextracted lipid or oil is determinable by analysis by using gaschromatography of fatty acid methyl esters prepared from the extractedlipid or oil.

In yet another embodiment, the process further comprises harvesting thepart from a plant.

In an embodiment, the plant part is a vegetative plant part whichcomprises a total non-polar lipid content of at least about 18%, atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, between 18% and 75%, between about 20% and 75%, between about 30%and 75%, between about 40% and 75%, between about 50% and 75%, betweenabout 60% and 75%, or between about 25% and 50% (w/w dry weight).

In a further embodiment, the plant part is a vegetative plant part whichcomprises a total TAG content of at least about 18%, at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 55%,at least about 60%, at least about 65%, at least about 70%, between 18%and 75%, between about 20% and 75%, between about 30% and 75%, betweenabout 40% and 75%, between about 50% and 75%, between about 60% and 75%,or between about 25% and 50% (w/w dry weight).

In another embodiment, the plant part is a vegetative plant part whichcomprises a total non-polar lipid content of at least about 11%, atleast about 12%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, between 8% and 75%, between10% and 75%, between 11% and 75%, between about 15% and 75%, betweenabout 20% and 75%, between about 30% and 75%, between about 40% and 75%,between about 50% and 75%, between about 60% and 75%, or between about25% and 50% (w/w dry weight), and wherein the vegetative plant part isfrom a 16:3 plant.

In yet another embodiment, the plant part is a vegetative plant partwhich comprises a total TAG content of at least about 11%, at leastabout 12%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, between 8% and 75%, between 10%and 75%, between 11% and 75%, between about 15% and 75%, between about20% and 75%, between about 30% and 75%, between about 40% and 75%,between about 50% and 75%, between about 60% and 75%, or between about25% and 50% (w/w dry weight), and wherein the vegetative plant part isfrom a 16:3 plant.

Also provided is a process for producing seed, the process comprising:

i) growing a plant of the invention, and

ii) harvesting seed from the plant.

In an embodiment, the above process comprises growing a population of atleast about 1000 plants, each being a plant of the invention, andharvesting seed from the population of plants.

In yet a further aspect, the present invention provides a fermentationprocess comprising the steps of:

i) providing a vessel containing a liquid composition comprising arecombinant eukaryotic cell of the invention, or the transgenicnon-human organism of the invention, wherein the cell or non-humanorganism is suitable for fermentation, and constituents required forfermentation and fatty acid biosynthesis, and

ii) providing conditions conducive to the fermentation of the liquidcomposition contained in said vessel.

Also provided is recovered or extracted lipid obtainable from arecombinant eukaryotic cell of the invention, a transgenic non-humanorganism or a part thereof of the invention, a transgenic plant or partthereof of the invention, a seed of the invention, or a transgenic cellor transgenic plant or part thereof of the invention, or obtainable bythe process of the invention.

In a further aspect, the present invention provides an industrialproduct produced by the process of the invention, which is a hydrocarbonproduct such as fatty acid esters, preferably fatty acid methyl estersand/or a fatty acid ethyl esters, an alkane such as methane, ethane or alonger-chain alkane, a mixture of longer chain alkanes, an alkene, abiofuel, carbon monoxide and/or hydrogen gas, a bioalcohol such asethanol, propanol, or butanol, biochar, or a combination of carbonmonoxide, hydrogen and biochar.

Also provided is the use of a recombinant eukaryotic cell of theinvention, a transgenic non-human organism or a part thereof of theinvention, a transgenic plant or part thereof of the invention, a seedof the invention, or a transgenic cell or transgenic plant or partthereof of the invention, or the recovered or extracted lipid of theinvention for the manufacture of an industrial product.

Examples of industrial products of the invention include, but are notlimited to, a hydrocarbon product such as fatty acid esters, preferablyfatty acid methyl esters and/or a fatty acid ethyl esters, an alkanesuch as methane, ethane or a longer-chain alkane, a mixture of longerchain alkanes, an alkene, a biofuel, carbon monoxide and/or hydrogengas, a bioalcohol such as ethanol, propanol, or butanol, biochar, or acombination of carbon monoxide, hydrogen and biochar.

In a further aspect, the present invention provides a process forproducing fuel, the process comprising:

i) reacting the lipid of the invention with an alcohol, optionally, inthe presence of a catalyst, to produce alkyl esters, and

ii) optionally, blending the alkyl esters with petroleum based fuel.

In an embodiment of the above process, the alkyl esters are methylesters.

In yet a further aspect, the present invention provides a process forproducing a synthetic diesel fuel, the process comprising:

i) converting the lipid in a recombinant eukaryotic cell of theinvention, a transgenic non-human organism or a part thereof of theinvention, a transgenic plant or part thereof of the invention, a seedof the invention, or a transgenic cell or transgenic plant or partthereof of the invention, to a bio-oil by a process comprising pyrolysisor hydrothermal processing or to a syngas by gasification, and

ii) converting the bio-oil to synthetic diesel fuel by a processcomprising fractionation, preferably selecting hydrocarbon compoundswhich condense between about 150° C. to about 200° C. or between about200° C. to about 300° C., or converting the syngas to a biofuel using ametal catalyst or a microbial catalyst.

In another aspect, the present invention provides a process forproducing a biofuel, the process comprising converting the lipid in arecombinant eukaryotic cell of the invention, a transgenic non-humanorganism or a part thereof of the invention, a transgenic plant or partthereof of the invention, a seed of the invention, or a transgenic cellor transgenic plant or part thereof of the invention to bio-oil bypyrolysis, a bioalcohol by fermentation, or a biogas by gasification oranaerobic digestion.

In an embodiment of the above process, the part is a vegetative plantpart.

Also provided is a process for producing a feedstuff, the processcomprising admixing a recombinant eukaryotic cell of the invention, atransgenic non-human organism or a part thereof of the invention, atransgenic plant or part thereof of the invention, a seed of theinvention, or a transgenic cell or transgenic plant or part thereof ofthe invention, or obtainable by the process of the invention, or anextract or portion thereof, with at least one other food ingredient.

In a further aspect, the present invention provides feedstuffs,cosmetics or chemicals comprising a recombinant eukaryotic cell of theinvention, a transgenic non-human organism or a part thereof of theinvention, a transgenic plant or part thereof of the invention, a seedof the invention, or a transgenic cell or transgenic plant or partthereof of the invention, or obtainable by the process of the invention,or an extract or portion thereof.

In another aspect, the present invention provides a process for feedingan animal, the process comprising providing to the animal the transgenicplant or part thereof of the invention, a seed of the invention, ortransgenic plant or part thereof of the invention, or the recovered orextracted lipid of the invention.

Any embodiment herein shall be taken to apply mutatis mutandis to anyother embodiment unless specifically stated otherwise.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended for the purpose ofexemplification only.

Functionally-equivalent products, compositions and methods are clearlywithin the scope of the invention, as described herein.

Throughout this specification, unless specifically stated otherwise orthe context requires otherwise, reference to a single step, compositionof matter, group of steps or group of compositions of matter shall betaken to encompass one and a plurality (i.e. one or more) of thosesteps, compositions of matter, groups of steps or group of compositionsof matter.

The invention is hereinafter described by way of the followingnon-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1. A representation of lipid synthesis in eukaryotic cells, showingexport of some of the fatty acids synthesized in the plastids to theEndoplasmic Reticulum (ER) via the Plastid Associated Membrane (PLAM),and import of some of the fatty acids into the plastid from the ER foreukaryotic galactolipid synthesis. Abbreviations:

-   -   Acetyl-CoA and Malonyl-CoA: acetyl-coenzyme A and        malonyl-coenzymeA;    -   ACCase: Acetyl-CoA carboxylase;    -   FAS: fatty acid synthase complex;    -   16:0-ACP, 18:0-ACP and 18:1-ACP: C16:0-acyl carrier protein        (ACP), C18:0-acyl carrier protein, C18:1-acyl carrier protein;    -   KAS II: ketoacyl-ACP synthase II (EC 2.3.1.41);    -   PLPAAT: plastidial LPAAT;    -   PGPAT: plastidial GPAT;    -   PAP: PA phosphorylase (EC 3.1.3.4);    -   G3P: glycerol-3-phosphate;    -   LPA: lysophosphatidic acid;    -   PA: phosphatidic acid;    -   DAG: diacylglycerol;    -   TAG: triacylglycerol;    -   Acyl-CoA and Acyl-PC: acyl-coenzyme A and        acyl-phosphatidylcholine;    -   PC: phosphatidylcholine;    -   GPAT: glycerol-3-phosphate acyltransferase;    -   LPAAT: lysophosphatidic acid acyltransferase (EC 2.3.1.51);    -   LPCAT: acyl-CoA:lysophosphatidylcholine acyltransferase; or        synonyms 1-acylglycerophosphocholine O-acyltransferase;        acyl-CoA:1-acyl-sn-glyccro-3-phosphocholine O-acyltransferase        (EC 2.3.1.23);    -   CPT: CDP-choline:diacylglycerol cholinephosphotransferase; or        synonyms 1-alkyl-2-acetylglycerol cholinephosphotransferase;        alkylacylglycerol cholinephosphotransferase;        cholinephosphotransferase; phosphorylcholine-glyceride        transferase (EC 2.7.8.2);    -   PDCT: phosphatidylcholine:diacylglycerol        cholinephosphotransferase;    -   PLC: phospholipase C (EC 3.1.4.3);    -   PLD: Phospholipase D; choline phosphatase; lecithinase D;        lipophosphodiesterase II (EC 3.1.4.4);    -   PDAT: phospholipid:diacylglycerol acyltransferase; or synonym        phospholipid:1,2-diacyl-sn-glycerol O-acyltransferase (EC        2.3.1.158);    -   FAD2: fatty acid Δ12-desaturase; FAD3, fatty acid        Δ15-desaturase;    -   UDP-Gal: Uridine diphosphate galactose;    -   MGDS: monogalactosyldiacylglycerol synthase;    -   MGDG: monogalactosyldiacylglycerol; DGDG:        digalactosyldiacylglycerol    -   FAD6, 7, 8: plastidial fatty acid Δ112-desaturase, plastidial        ω3-desaturase, plastidial ω3-desaturase induced at low        temperature, respectively.

FIG. 2. Schematic genetic map of construct to increase seed oil contentin dicotyledonous plants. Abbreviations: PRO Pissa-Vicilin, Pisumsativum vicilin promoter and 5′ UTR; TMV leader, 5′UTR of tobacco mosaicvirus; Arath-DGAT1, protein coding region encoding A. thaliana DGAT1;TER Glyma-Lectin, 3′ terminator/polyadenylation region of a G. maxlectin gene; PRO Phavu-Phaseolin, promoter from a Phaseolus vulgarisphaseolin protein gene; Arath-WRI1, protein coding region encoding A.thaliana WRI1; TER Agrtu-NOS, 3′ terminator/polyadenylation region of anAgrobacterium tumefaciens Nos gene; PRO Phavu-PHA, promoter of aPhaseolus vulgaris phaseolin gene; Sesin-Oleosin, protein coding regionencoding a Sesame indicum oleosin gene; TER Phavu-PHA, 3′terminator/polyadenylation region of a Phaseolus vulgaris phaseolingene.

FIG. 3. Schematic diagram of vector pOIL122. Abbreviations: TERAgrtu-Nos, Agrobacterium tumefaciens nopaline synthase terminator,NPTII, neomycin phosphotransferase protein coding region; PROCaMV35S-Ex2, Cauliflower Mosaic Virus 35S promoter with double enhancerregion; Arath-DGAT1, Arabidopsis thaliana DGAT1 acyltransferase proteincoding region; PRO Arath-Rubisco SSU, A. thaliana Rubisco small subunitpromoter; Arath-FATA2, A. thaliana FATA2 thioesterase protein codingregion; Arath-WRI, A. thaliana WRI1 transcription factor protein codingregion; TER Glyma-Lectin, Glycine max lectin terminator, enTCUP2promoter, Nicotiana tabacum cryptic constitutive promoter; attB1 andattB2, Gateway recombination sites; NB SDP1 fragment, Nicotianabenthamiana SDP1 region targeted for hpRNAi silencing; OCS terminator,A. tumefaciens octopine synthase terminator. Backbone features outsidethe T-DNA region are derived from pORE04 (Coutu et al., 2007).

FIG. 4. Total fatty acid methyl ester (FAME) profiles (weight %)illustrating the effect of WRI1+DGAT1-mediated high oil background onMCFA production in Nicotiana benthamiana leaf (n=4). Highest MCFAproduction was observed after the addition of Arath-WRI1.

FIG. 5. Leaf total FAME profiles (weight %) elucidating the effect ofWRI1 on MCFA accumulation (n=4). Addition of Arath-WRI1 greatlyincreased the production of the relevant fatty acid (C12:0, C14:0 orC16:0) relative to the previous addition of Cocnu-LPAAT alone.

FIG. 6. TFA levels (% weight), TAG levels, levels of MCFA (C16:0 andC14:0, % of total fatty acids) in TFA and MCFA in TAG (% of total fattyacid content in TAG) in plant cells after expression of combinations ofthree oil palm DGATs with FATB, LPAAT and WRI1. Numbers 1-10 are aslisted in the text (Example 9).

FIG. 7. TAG levels (% leaf dry weight) in N. benthamiana leaf tissue,infiltrated with genes encoding different WRI1 polypeptides either with(right hand bars) or without (left hand bars) co-expression of DGAT1(n=3). All samples were infiltrated with the P19 construct as well.

FIG. 8. Schematic representation of the N. benthamiana SDP1 hairpinconstruct. The genetic segments shown are as described in Example 11.Abbreviations are as for FIG. 3. attB sites represent recombinationsites from the pHELLSGATE12 vector.

FIG. 9. TAG content in green leaf samples of tobacco plants transformedwith the T-DNA from pOIL51, lines #61 and #69, harvested beforeflowering. The controls (parent) samples were from plants transformedwith the T-DNA from pJP3502.

FIG. 10. TAG levels (% dry weight) in root and stem tissue of wild-type(wt) and transgenic N. tabacum plants containing the T-DNA from pJP3502alone or additionally with the T-DNA from pOIL051.

FIG. 11. TAG levels (% dry weight) in root and stem tissue of wild-type(wt) and transgenic N. tabacum plants containing the T-DNA from pJP3502alone or additionally with the T-DNA from pOIL049.

FIG. 12. TAG content in leaf samples of transformed tobacco plants atseed-setting stage of growth, transformed with the T-DNA from pOIL049,lines #23c and #32b. The controls (parent) samples were from plantstransformed with the T-DNA from pJP3502. The upper line shows 18:2percentage in the TAG and the lower line shows the 18:3 (ALA) percentagein the fatty acid content.

FIG. 13. A. Starch content in leaf tissue from wild-type plants (WT) andtransgenic plants containing the T-DNA from pJP3502 (HO control) or theT-DNAs from both pJP3502 and pOIL051 (pOIL51.61 and pOIL51.69) or bothpJP3502 and pOIL049 (pOIL49.32b). Data represent combined results fromat least three individual plants. B. Correlation between starch and TAGcontent in leaf tissue of wild-type plants (WT) and transgenic plantscontaining the T-DNA from pJP3502 (HO control) or T-DNAs from bothpJP3502 and pOIL051 (pOIL51.61 and pOIL51.69) or both pJP3502 andpOIL049 (pOIL49.32b). Data represent combined results from at leastthree individual plants.

FIG. 14. Schematic representation of the pTV55 binary vector.Abbreviations: PRO, promoter; TER, 3′ termination/polyadenylationregion; Arath, A. thaliana; Linus, Linum usitatissimum; Nicta, Nicotianatabacum; Glyma, G. max; Cnl1, conlinin 1 from flax; Cnl2, Conlinin 2from flax; MAR Nicat-RB7, matrix attachment region from the tobacco RB7,or as in FIG. 3. Gene abbreviations MGAT2, DGAT1, GPAT4, WRI1 as in thetext.

FIG. 15. Oil content (%) of C. sativa T2 seeds transformed with pTV55,pTV56 and pTV57 as determined by NMR. Each data point represents theaverage oil content of three independent batches of 50 mg seed for eachtransgenic line. Negative control seeds were wild-type (untransformed)C. sativa seeds, grown under the same conditions in the greenhouse. Nindicates the number of independent transgenic events for eachconstruct.

FIG. 16. Phylogenetic tree of LDAP polypeptides (Example 15).

FIG. 17. Schematic representation of the genetic construct pJP3506including the T-DNA region between the left and right borders.Abbreviations are as for FIG. 3 and: Sesin-Oleosin, Sesame indicumoleosin protein coding region.

FIG. 18. Yield and calorific value changes for bio-oil production by HTPof wild-type and transgenic, high oil tobacco vegetative plant materialas feedstock.

KEY TO THE SEQUENCE LISTING

-   SEQ ID NO:1 Arabidopsis thaliana DGAT1 polypeptide (CAB44774.1)-   SEQ ID NO:2 Arabidopsis thaliana DGAT2 polypeptide (NP_566952.1)-   SEQ ID NO:3 Ricinus communis DGAT2 polypeptide (AAY16324.1)-   SEQ ID NO:4 Vernicia fordii DGAT2 polypeptide (ABC94474.1)-   SEQ ID NO:5 Mortierella ramanniana DGAT2 polypeptide (AAK84179.1)-   SEQ ID NO:6 Homo sapiens DGAT2 polypeptide (Q96PD7.2)-   SEQ ID NO:7 Homo sapiens DGAT2 polypeptide (Q58HT5.1)-   SEQ ID NO:8 Bos taurus DGAT2 polypeptide (Q70VZ8.1)-   SEQ ID NO:9 Mus musculus DGAT2 polypeptide (AAK84175.1)-   SEQ ID NO:10 YFP tripeptide—conserved DGAT2 and/or MGAT1/2 sequence    motif-   SEQ ID NO:11 HPHG tetrapeptide—conserved DGAT2 and/or MGAT1/2    sequence motif-   SEQ ID NO:12 EPHS tetrapeptide—conserved plant DGAT2 sequence motif-   SEQ ID NO:13 RXGFX(K/R)XAXXXGXXX(L/V)VPXXXFG(E/Q)—long conserved    sequence motif of DGAT2 which is part of the putative glycerol    phospholipid domain-   SEQ ID NO:14 FLXLXXXN—conserved sequence motif of mouse DGAT2 and    MGAT1/2 which is a putative neutral lipid binding domain-   SEQ ID NO:15 plsC acyltransferase domain (PF01553) of GPAT-   SEQ ID NO:16 HAD-like hydrolase (PF12710) superfamily domain of GPAT-   SEQ ID NO:17 Phosphoserine phosphatase domain (PF00702). GPAT4-8    contain a N-terminal region homologous to this domain-   SEQ ID NO: 18 Conserved GPAT amino acid sequence GDLVICPEGTTCREP-   SEQ ID NO:19 Conserved GPAT/phosphatase amino acid sequence (Motif    I)-   SEQ ID NO:20 Conserved GPAT/phosphatase amino acid sequence (Motif    III)-   SEQ ID NO:21 Arabidopsis thaliana WRI1 polypeptide (A8MS57)-   SEQ ID NO:22 Arabidopsis thaliana WRI1 polypeptide (Q6X5Y6)-   SEQ ID NO:23 Arabidopsis lyrata subsp. lyrata WRI1 polypeptide    (XP_002876251.1)-   SEQ ID NO:24 Brassica napus WRI1 polypeptide (ABD16282.1)-   SEQ ID NO:25 Brassica napus WRI1 polypeptide (ADO16346.1)-   SEQ ID NO:26 Glycine max WRI1 polypeptide (XP_003530370.1)-   SEQ ID NO:27 Jatropha curcas WRI1 polypeptide (AEO22131.1)-   SEQ ID NO:28 Ricinus communis WRI1 polypeptide (XP_02525305.1)-   SEQ ID NO:29 Populus trichocarpa WRI1 polypeptide (XP_002316459.1)-   SEQ ID NO:30 Vitis vinifera WRI1 polypeptide (CB129147.3)-   SEQ ID NO:31 Brachypodium distachyon WRI1 polypeptide    (XP_003578997.1)-   SEQ ID NO:32 Hordeum vulgare subsp. vulgare WRI1 polypeptide    (BAJ86627.1)-   SEQ ID NO:33 Oryza sativa WRI1 polypeptide (EAY79792.1)-   SEQ ID NO:34 Sorghum bicolor WRI1 polypeptide (XP_002450194.1)-   SEQ ID NO:35 Zea mays WRI1 polypeptide (ACG32367.1)-   SEQ ID NO:36 Brachypodium distachyon WRI1 polypeptide    (XP_003561189.1)-   SEQ ID NO:37 Brachypodium sylvaticum WRI1 polypeptide (ABL85061.1)-   SEQ ID NO:38 Oryza sativa WRI1 polypeptide (BAD68417.1)-   SEQ ID NO:39 Sorghum bicolor WRI1 polypeptide (XP_002437819.1)-   SEQ ID NO:40 Sorghum bicolor WRI1 polypeptide (XP_002441444.1)-   SEQ ID NO:41 Glycine max WRI1 polypeptide (XP_003530686.1)-   SEQ ID NO:42 Glycine max WRI1 polypeptide (XP_003553203.1)-   SEQ ID NO:43 Populus trichocarpa WRI1 polypeptide (XP 002315794.1)-   SEQ ID NO:44 Vitis vinifera WRI1 polypeptide (XP_002270149.1)-   SEQ ID NO:45 Glycine max WRI1 polypeptide (XP_003533548.1)-   SEQ ID NO:46 Glycine max WRI1 polypeptide (XP_003551723.1)-   SEQ ID NO:47 Medicago truncatula WRI1 polypeptide (XP_003621117.1)-   SEQ ID NO:48 Populus trichocarpa WRI1 polypeptide (XP_002323836.1)-   SEQ ID NO:49 Ricinus communis WRI1 polypeptide (XP_002517474.1)-   SEQ ID NO:50 Vitis vinifera WRI1 polypeptide (CAN79925.1)-   SEQ ID NO:51 Brachypodium distachyon WRI1 polypeptide    (XP_003572236.1)-   SEQ ID NO:52 Oryza sativa WRI1 polypeptide (BAD10030.1)-   SEQ ID NO:53 Sorghum bicolor WRI1 polypeptide (XP_002444429.1)-   SEQ ID NO:54 Zea mays WRI1 polypeptide (NP_001170359.1)-   SEQ ID NO:55 Arabidopsis lyrata subsp. lyrata WRI1 polypeptide    (XP_002889265.1)-   SEQ ID NO:56 Arabidopsis thaliana WRI1 polypeptide (AAF68121.1)-   SEQ ID NO:57 Arabidopsis thaliana WRI1 polypeptide (NP_178088.2)-   SEQ ID NO:58 Arabidopsis lyrata subsp. lyrata WRI1 polypeptide    (XP_002890145.1)-   SEQ ID NO:59 Thellungiella halophila WRI1 polypeptide (BAJ33872.1)-   SEQ ID NO:60 Arabidopsis thaliana WRI1 polypeptide (NP_563990.1)-   SEQ ID NO:61 Glycine max WRI1 polypeptide (XP_003530350.1)-   SEQ ID NO:62 Brachypodium distachyon WRI1 polypeptide    (XP_003578142.1)-   SEQ ID NO:63 Oryza sativa WRI1 polypeptide (EAZ09147.1)-   SEQ ID NO:64 Sorghum bicolor WRI1 polypeptide (XP_002460236.1)-   SEQ ID NO:65 Zea mays WRI1 polypeptide (NP_001146338.1)-   SEQ ID NO:66 Glycine max WRI1 polypeptide (XP_003519167.1)-   SEQ ID NO:67 Glycine max WRI1 polypeptide (XP_003550676.1)-   SEQ ID NO:68 Medicago truncatula WRI1 polypeptide (XP 003610261.1)-   SEQ ID NO:69 Glycine max WRI1 polypeptide (XP_003524030.1)-   SEQ ID NO:70 Glycine max WRI1 polypeptide (XP_003525949.1)-   SEQ ID NO:71 Populus trichocarpa WRI1 polypeptide (XP_002325111.1)-   SEQ ID NO:72 Vitis vinifera WRI1 polypeptide (CB136586.3)-   SEQ ID NO:73 Vitis vinifera WRI1 polypeptide (XP_002273046.2)-   SEQ ID NO:74 Populus trichocarpa WRI1 polypeptide (XP_002303866.1)-   SEQ ID NO:75 Vitis vinifera WRI1 polypeptide (CB125261.3)-   SEQ ID NO:76 Sorbi-WRL1-   SEQ ID NO: 77 Lupan-WRL1-   SEQ ID NO:78 Ricco-WRL1-   SEQ ID NO:79 Lupin angustifolius WRI1 polypeptide-   SEQ ID NO:80 Aspergillus fumigatus DGAT1 polypeptide (XP_755172.1)-   SEQ ID NO:81 Ricinus communis DGAT1 polypeptide (AAR11479.1)-   SEQ ID NO:82 Vernicia fordii DGAT1 polypeptide (ABC94472.1)-   SEQ ID NO:83 Vernonia galamensis DGAT1 polypeptide (ABV21945.1)-   SEQ ID NO:84 Vernonia galamensis DGAT1 polypeptide (ABV21946.1)-   SEQ ID NO:85 Euonymus alatus DGAT1 polypeptide (AAV31083.1)-   SEQ ID NO:86 Caenorhabditis elegans DGAT1 polypeptide (AAF82410.1)-   SEQ ID NO:87 Rattus norvegicus DGAT1 polypeptide (NP_445889.1)-   SEQ ID NO:88 Homo sapiens DGAT1 polypeptide (NP_036211.2)-   SEQ ID NO:89 WRI1 motif (R G V T/S R H R W T G R)-   SEQ ID NO:90 WRI1 motif (F/Y E A H L W D K)-   SEQ ID NO:91 WRI1 motif (D L A A L K Y W G)-   SEQ ID NO:92 WRI1 motif (S X G F S/A R G X)-   SEQ ID NO:93 WRI1 motif (H H H/Q N G R/K W E A R I G R/K V)-   SEQ ID NO:94 WRI1 motif (Q E E A A A X Y D)-   SEQ ID NO:95 Brassica napus oleosin polypeptide (CAA57545.1)-   SEQ ID NO:96 Brassica napus oleosin S1-1 polypeptide (ACG69504.1)-   SEQ ID NO:97 Brassica napus oleosin S2-1 polypeptide (ACG69503.1)-   SEQ ID NO:98 Brassica napus oleosin S3-1 polypeptide (ACG69513.1)-   SEQ ID NO:99 Brassica napus oleosin S4-1 polypeptide (ACG69507.1)-   SEQ ID NO:100 Brassica napus oleosin S5-1 polypeptide (ACG69511.1)-   SEQ ID NO:101 Arachis hypogaea oleosin 1 polypeptide (AAZ20276.1)-   SEQ ID NO: 102 Arachis hypogaea oleosin 2 polypeptide (AAU21500.1)-   SEQ ID NO:103 Arachis hypogaea oleosin 3 polypeptide (AAU21501.1)-   SEQ ID NO:104 Arachis hypogaea oleosin 5 polypeptide (ABC96763.1)-   SEQ ID NO:105 Ricinus communis oleosin 1 polypeptide (EEF40948.1)-   SEQ ID NO:106 Ricinus communis oleosin 2 polypeptide (EEF51616.1)-   SEQ ID NO: 107 Glycine max oleosin isoform a polypeptide (P29530.2)-   SEQ ID NO:108 Glycine max oleosin isoform b polypeptide (P29531.1)-   SEQ ID NO:109 Linum usitalissimum oleosin low molecular weight    isoform polypeptide (ABB01622.1)-   SEQ ID NO:110 amino acid sequence of Linum usitatissimum oleosin    high molecular weight isoform polypeptide (ABB01624.1)-   SEQ ID NO:111 Helianthus annuus oleosin polypeptide (CAA44224.1)-   SEQ ID NO: 112 Zea mays oleosin polypeptide (NP_001105338.1)-   SEQ ID NO:113 Brassica napus steroleosin polypeptide (ABM30178.1)-   SEQ ID NO:114 Brassica napus steroleosin SLO1-1 polypeptide    (ACG69522.1)-   SEQ ID NO:115 Brassica napus steroleosin SLO2-1 polypeptide    (ACG69525.1)-   SEQ ID NO: 116 Sesamum indicum steroleosin polypeptide (AAL13315.1)-   SEQ ID NO:117 Zea mays steroleosin polypeptide (NP 001152614.1)-   SEQ ID NO:118 Brassica napus caleosin CLO-1 polypeptide (ACG69529.1)-   SEQ ID NO: 119 Brassica napus caleosin CLO-3 polypeptide    (ACG69527.1)-   SEQ ID NO:120 Sesamum indicum caleosin polypeptide (AAF13743.1)-   SEQ ID NO: 121 Zea mays caleosin polypeptide (NP 001151906.1)-   SEQ ID NO:122 pJP3502 TDNA (inserted into genome) sequence-   SEQ ID NO:123 pJP3507 vector sequence-   SEQ ID NO:124 Linker sequence-   SEQ ID NO: 125 Partial Nicotiana benthamiana CGI-58 sequence    selected for hpRNAi silencing (pTV46)-   SEQ ID NO:126 Partial N. tabacum AGPase sequence selected for hpRNAi    silencing (pTV35)-   SEQ ID NO:127 GXSXG lipase motif-   SEQ ID NO:128 HX(4)D acyltransferase motif-   SEQ ID NO:129 VX(3)HGF probable lipid binding motif-   SEQ ID NO:130 Arabidopsis thaliana CGi58 polynucleotide    (NM_118548.1)-   SEQ ID NO:131 Brachypodium distachyon CGi58 polynucleotide    (XM_003578402.1)-   SEQ ID NO:132 Glycine max CGi58 polynucleotide (XM_003523590.1)-   SEQ ID NO:133 Zea mays CGi58 polynucleotide (NM_001155541.1)-   SEQ ID NO: 134 Sorghum bicolor CGi58 polynucleotide (XM_002460493.1)-   SEQ ID NO:135 Ricinus communis CGi58 polynucleotide (XM_002510439.1)-   SEQ ID NO:136 Medicago truncalula CGi58 polynucleotide    (XM_003603685.1)-   SEQ ID NO:137 Arabidopsis thaliana LEC2 polynucleotide (NM_102595.2)-   SEQ ID NO:138 Medicago truncatula LEC2 polynucelotide (X60387.1)-   SEQ ID NO:139 Brassica napus LEC2 polynucelotide (HM370539.1)-   SEQ ID NO:140 Arabidopsis thaliana BBM polynucleotide (NM_121749.2)-   SEQ ID NO:141 Medicago truncatula BBM polynucleotide (AY899909.1)-   SEQ ID NO: 142 Arabidopsis thaliana LEC2 polypeptide (NP_564304.1)-   SEQ ID NO:143 Medicago truncatula LEC2 polypeptide (CAA42938.1)-   SEQ ID NO: 144 Brassica napus LEC2 polypeptide (ADO16343.1)-   SEQ ID NO: 145 Arabidopsis thaliana BBM polypeptide (NP_197245.2)-   SEQ ID NO:146 Medicago truncatula BBM polypeptide (AAW82334.1)-   SEQ ID NO:147 Inducible Aspergillus niger alcA promoter-   SEQ ID NO:148 AlcR inducer that activates the AlcA promotor in the    presence of ethanol-   SEQ ID NO:149 Arabidopsis thaliana LEC1; (AAC39488)-   SEQ ID NO:150 Arabidopsis lyrata LEC1 (XP_002862657)-   SEQ ID NO:151 Brassica napus LEC1 (ADF81045)-   SEQ ID NO: 152 Ricinus communis LEC1 (XP_002522740)-   SEQ ID NO:153 Glycine max LEC1 (XP_006582823)-   SEQ ID NO: 154 Medicago truncatula LEC1 (AFK49653)-   SEQ ID NO: 155 Zea mays LEC1 (AAK95562)-   SEQ ID NO:156 Arachis hypogaea LEC1 (ADC33213)-   SEQ ID NO: 157 Arabidopsis thaliana LEC1-like (AAI15924)-   SEQ ID NO:158 Brassica napus LEC1-like (AH194922)-   SEQ ID NO: 159 Phaseolus coccineus LEC1-like (AAN01148)-   SEQ ID NO:160 Arabidopsis thaliana FUS3 (AAC35247)-   SEQ ID NO:161 Brassica napus FUS3-   SEQ ID NO:162 Medicago truncalula FUS3-   SEQ ID NO:163 Arabidopsis thaliana SDP1 cDNA sequence, Accession No.    NM_120486, 3275 nt-   SEQ ID NO:164 Brassica napus SDP1 cDNA; Accession No. GN078290-   SEQ ID NO:165 Brachypodium distachyon SDP1 cDNA, 2670 nt-   SEQ ID NO:166 Populus trichocarpa SDP1 cDNA, 3884 nt-   SEQ ID NO: 167 Medicago truncatula SDP1 cDNA; XM_003591377; 2490 nt-   SEQ ID NO:168 Glycine max SDP1 cDNA XM_003521103; 2783 nt-   SEQ ID NO:169 Sorghum bicolor SDP1 cDNA XM_002458486; 2724 nt-   SEQ ID NO:170 Zea mays SDP1 cDNA, NM_001175206; 2985 nt-   SEQ ID NO:171 Physcomitrella patens SDP1 cDNA, XM_001758117; 1998 nt-   SEQ ID NO:172 Hordeum vulgare SDP1 cDNA, AK372092; 3439 nt-   SEQ ID NO:173 Nicotiana benthamiana SDP1 cDNA, Nbv5 tr6404201-   SEQ ID NO:174 Nicotiana benthamiana SDP1 cDNA region targeted for    hpRNAi silencing-   SEQ ID NO: 175 Promoter of Arabidopsis thaliana SDP1 gene, 1.5 kb-   SEQ ID NO:176 Nucleotide sequence of the complement of the    pSSU-Oleosin gene in the T-DNA of pJP3502. In order (complementary    sequences): Glycine max Lectin terminator 348 nt, 3′ exon 255 nt,    UBQ10 intron 304 nt, 5′ exon 213 nt, SSU promoter 1751 nt-   SEQ ID NO:177 Arabidopsis thaliana plastidial GPAT cDNA, NM_179407-   SEQ ID NO:178 Arabidopsis thaliana plastidial GPAT polypeptide,    NM_179407-   SEQ ID NO:179 Populus trichocarpa plastidial GPAT cDNA, XP_006368351-   SEQ ID NO:180 Jatropha curcas plastidial GPAT cDNA, ACR61638-   SEQ ID NO:181 Ricinus communis plastidial GPAT cDNA, XP_002518993-   SEQ ID NO:182 Helianthus annuus plastidial GPAT cDNA, ADV16382-   SEQ ID NO:183 Medicago truncatula plastidial GPAT cDNA, XP_003612801-   SEQ ID NO:184 Glycine max plastidial GPAT cDNA, XP_003516958-   SEQ ID NO: 185 Carthamus tinctorius plastidial GPAT cDNA, CAHG3PACTR-   SEQ ID NO: 186 Solanum tuberosum plastidial GPAT cDNA, XP_006352898-   SEQ ID NO:187 Oryza sativa, Japonica plastidial GPAT cDNA,    NM_001072027-   SEQ ID NO: 188 Sorghum bicolor plastidial GPAT cDNA, XM_002467381-   SEQ ID NO:189 Zea mays plastidial GPAT cDNA, NM_001158637-   SEQ ID NO:190 Hordeum vulgare plastidial GPAT cDNA, AK371419-   SEQ ID NO: 191 Physcomitrella patens plastidial GPAT cDNA, XM    001771247-   SEQ ID NO: 192 Chlamydomonas reinhardtii plastidial GPAT cDNA,    XM_001694925-   SEQ ID NO:193 Cinnamomum camphora 14:0-ACP thioesterase (Cinca-TE),    chloroplastic, 382aa, (Accession No. Q39473.1)-   SEQ ID NO:194 Cocos nucifera acyl-ACP thioesterase FatB1 (Cocnu-TE1;    417aa, Accession No. AEM72519.1-   SEQ ID NO:195 Cocos nucifera acyl-ACP thioesterase FatB2 (Cocnu-TE2;    423aa, Accession No. AEM72520.1)-   SEQ ID NO:196 Cocos nucifera acyl-ACP thioesterase FatB3 (Cocnu-TE3;    414aa, Accession No. AEM72521.1)-   SEQ ID NO:197 Cuphea lanceolata acyl-(ACP) thioesterase type B    (Cupla-TE, 419aa, Accession No. CAB60830.1)-   SEQ ID NO:198 Cuphea viscosissima FatB1 (Cupvi-TE; 419aa, Accession    No. AEM72522.1)-   SEQ ID NO:199 Umbellularia californica 12:0-ACP thioesterase    (Lauroyl-acyl carrier protein thioesterase) (Umbca-TE, 382aa;    Accession No. Q41635.1)-   SEQ ID NO:200 Cocos nucifera LPAAT (Cocnu-LPAAT, 308aa, Accession    No. Q42670.1)-   SEQ ID NO:201 Arabidopsis thaliana plastidial LPAAT1 (Arath-PLPAAT;    356aa, Accession No. AEE85783.1)-   SEQ ID NO:202 Arabidopsis thaliana FATA1-   SEQ ID NO:203 Arabidopsis thaliana FATA2-   SEQ ID NO:204 Arabidopsis thaliana FATB-   SEQ ID NO:205 Arabidopsis thaliana WRI3-   SEQ ID NO:206 Arabidopsis thaliana WRI4-   SEQ ID NO:207 Avena sativa WRI1-   SEQ ID NO:208 Sorghum bicolor WRI1-   SEQ ID NO:209 Zea mays WRI1-   SEQ ID NO:210 Triadica sebifera WRI1-   SEQ ID NO:211 S. tuberosum Patatin B33 promoter sequence-   SEQ ID NOs 212 to 215 and 245 to 254 Oligonucleotide primers-   SEQ ID NO:216 Z. mays SEE1 promoter region (1970 nt from Accession    number AJ494982)-   SEQ ID NO:217 A. littoralis AlSAP promoter sequence, Accession No    DQ885219-   SEQ ID NO:218 A. rhizogenes ArRo1C promoter sequence, Accession No.    DQ160187-   SEQ ID NO:219 hpRNAi construct containing a 732 bp fragment of N.    benthamiana plastidial GPAT-   SEQ ID NO:220 Elaeis guineensis (oil palm) DGAT1-   SEQ ID NO:221 G. max MYB73, Accession No. ABH02868-   SEQ ID NO:222 A. thaliana bZIP53, Accession No. AAM14360-   SEQ ID NO:223 A. thaliana AGL15, Accession No NP_196883-   SEQ ID NO:224 A. thaliana MYB118, Accession No. AAS58517-   SEQ ID NO:225 A. thaliana MYB115, Accession No. AAS10103-   SEQ ID NO:226 A. thaliana TANMEI, Accession No. BAE44475-   SEQ ID NO:227 A. thaliana WUS, Accession No. NP_565429-   SEQ ID NO:228 B. napus GFR2a1, Accession No. AFB74090-   SEQ ID NO:229 B. napus GFR2a2, Accession No. AFB74089-   SEQ ID NO:230 A. thaliana PHR1, Accession No. AAN72198-   SEQ ID NO:231 N. benthamiana TGD1 fragment-   SEQ ID NO:232 Potato SDP1 amino acid-   SEQ ID NO:233 Potato SDP1 nucleotide sequence-   SEQ ID NO:234 Potato AGPase small subunit-   SEQ ID NO:235 Potato AGPase small subunit nucleotide sequence:-   SEQ ID NO:236 Sapium sebiferum LDAP-1 nucleotide sequence-   SEQ ID NO:237 Sapium sebiferum LDAP-amino acid sequence-   SEQ ID NO:238 Sapium sebiferum LDAP-2 nucleotide sequence-   SEQ ID NO:239 Sapium sebiferum LDAP-2 amino acid sequence-   SEQ ID NO:240 Sapium sebiferum LDAP-3 nucleotide sequence-   SEQ ID NO:241 Sapium sebiferum LDAP-3 amino acid sequence-   SEQ ID NO:242 S. bicolor SDP1 (accession number XM_002463620)-   SEQ ID NO:243 T. aeslivum SDP1 nucleotide sequence (Accession number    AK334547)-   SEQ ID NO:244 S. bicolor SDP1 hpRNAi fragment

DETAILED DESCRIPTION OF THE INVENTION General Techniques

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (e.g., in cell culture,molecular genetics, plant biology, cell biology, protein chemistry,lipid and fatty acid chemistry, biofeul production, and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, andimmunological techniques utilized in the present invention are standardprocedures, well known to those skilled in the art. Such techniques aredescribed and explained throughout the literature in sources such as, J.Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons(1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory Press (1989), T. A. Brown (editor), EssentialMolecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press(1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A PracticalApproach, Volumes 1-4, IRL Press (1995 and 1996), F. M. Ausubel et al.(editors), Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience (1988, including all updates untilpresent), Ed Harlow and David Lane (editors) Antibodies: A LaboratoryManual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al.(editors) Current Protocols in Immunology, John Wiley & Sons (includingall updates until present).

Selected Definitions

The term “transgenic non-human organism” refers to, for example, a wholeplant, alga, non-human animal, or an organism suitable for fermentationsuch as a yeast or fungus, comprising one or more exogenouspolynucleotides (transgene) or polypeptides. In an embodiment, thetransgenic non-human organism is not an animal or part thereof. In oneembodiment, the transgenic non-human organism is a phototrophic organism(for example, a plant or alga) capable of obtaining energy from sunlightto synthesize organic compounds for nutrition.

The term “exogenous” in the context of a polynucleotide or polypeptiderefers to the polynucleotide or polypeptide when present in a cell whichdoes not naturally comprise the polynucleotide or polypeptide. Such acell is referred to herein as a “recombinant cell” or a “transgeniccell”. In an embodiment, the exogenous polynucleotide or polypeptide isfrom a different genus to the cell comprising the exogenouspolynucleotide or polypeptide. In another embodiment, the exogenouspolynucleotide or polypeptide is from a different species. In oneembodiment the exogenous polynucleotide or polypeptide is expressed in ahost plant or plant cell and the exogenous polynucleotide or polypeptideis from a different species or genus. The exogenous polynucleotide orpolypeptide may be non-naturally occurring, such as for example, asynthetic DNA molecule which has been produced by recombinant DNAmethods. The DNA molecule may, often preferably, include a proteincoding region which has been codon-optimised for expression in the cell,thereby producing a polypeptide which has the same amino acid sequenceas a naturally occurring polypeptide, even though the nucleotidesequence of the protein coding region is non-naturally occurring. Theexogenous polynucleotide may encode, or the exogenous polypeptide maybe: a diacylglycerol acyltransferase (DGAT) such as a DGAT1 or a DGAT2,a Wrinkled 1 (WRI1) transcription factor, on OBC such as an Oleosin orpreferably an LDAP, a fatty acid thioesterase such as a FATA or FATBpolypeptide, or a silencing suppressor polypeptide.

As used herein, the term “extracted lipid” refers to a compositionextracted from a transgenic organism or part thereof which comprises atleast 60% (w/w) lipid.

As used herein, the term “non-polar lipid” refers to fatty acids andderivatives thereof which are soluble in organic solvents but insolublein water. The fatty acids may be free fatty acids and/or in anesterified form. Examples of esterified forms include, but are notlimited to, triacylglycerol (TAG), diacylyglycerol (DAG),monoacylglycerol (MAG). Non-polar lipids also include sterols, sterolesters and wax esters. Non-polar lipids are also known as “neutrallipids”. Non-polar lipid is typically a liquid at room temperature.Preferably, the non-polar lipid predominantly (>50%) comprises fattyacids that are at least 16 carbons in length. More preferably, at least50% of the total fatty acids in the non-polar lipid are C18 fatty acidsfor example, oleic acid. Preferably, at least 5% of the total fattyacids in the non-polar lipids are C12 or C14 fatty acids, or both. In anembodiment, at least 50%, more preferably at least 70%, more preferablyat least 80%, more preferably at least 90%, more preferably at least91%, more preferably at least 92%, more preferably at least 93%, morepreferably at least 94%, more preferably at least 95%, more preferablyat least 96%, more preferably at least 97%, more preferably at least98%, more preferably at least 99% of the fatty acids in non-polar lipidof the invention can be found as TAG. The non-polar lipid may be furtherpurified or treated, for example by hydrolysis with a strong base torelease the free fatty acid, or by fractionation, distillation, or thelike. Non-polar lipid may be present in or obtained from plant partssuch as seed, leaves, tubers, beets or fruit, from recombinant cells orfrom non-human organisms such as yeast. Non-polar lipid of the inventionmay form part of “seedoil” if it is obtained from seed.

The free and esterified sterol (for example, sitosterol, campesterol,stigmasterol, brassicasterol, Δ5-avenasterol, sitostanol, campestanol,and cholesterol) concentrations in the extracted lipid may be asdescribed in Phillips et al. (2002). Sterols in plant oils are presentas free alcohols, esters with fatty acids (esterified sterols),glycosides and acylated glycosides of sterols. Sterol concentrations innaturally occurring vegetable oils (seedoils) ranges up to a maximum ofabout 1100 mg/100 g. Hydrogenated palm oil has one of the lowestconcentrations of naturally occurring vegetable oils at about 60 mg/100g. The recovered or extracted seedoils of the invention preferably havebetween about 100 and about 1000 mg total sterol/100 g of oil. For useas food or feed, it is preferred that sterols are present primarily asfree or esterified forms rather than glycosylated forms. In the seedoilsof the present invention, preferably at least 50% of the sterols in theoils are present as esterified sterols, except for soybean seedoil whichhas about 25% of the sterols esterified. The canola seedoil and rapeseedoil of the invention preferably have between about 500 and about 800 mgtotal sterol/100 g, with sitosterol the main sterol and campesterol thenext most abundant. The corn seedoil of the invention preferably hasbetween about 600 and about 800 mg total sterol/100 g, with sitosterolthe main sterol. The soybean seedoil of the invention preferably hasbetween about 150 and about 350 mg total sterol/100 g, with sitosterolthe main sterol and stigmasterol the next most abundant, and with morefree sterol than esterified sterol. The cottonseed oil of the inventionpreferably has between about 200 and about 350 mg total sterol/100 g,with sitosterol the main sterol. The coconut oil and palm oil of theinvention preferably have between about 50 and about 100 mg totalsterol/100 g, with sitosterol the main sterol. The safflower seedoil ofthe invention preferably has between about 150 and about 250 mg totalsterol/100 g, with sitosterol the main sterol. The peanut seedoil of theinvention preferably has between about 100 and about 200 mg totalsterol/100 g, with sitosterol the main sterol. The sesame seedoil of theinvention preferably has between about 400 and about 600 mg totalsterol/100 g, with sitosterol the main sterol. The sunflower seedoil ofthe invention preferably has between about 200 and 400 mg totalsterol/100 g, with sitosterol the main sterol. Oils obtained fromvegetative plant parts according to the invention preferably have lessthan 200 mg total sterol/100 g, more preferably less than 100 mg. totalsterol/100 g, and most preferably less than 50 mg total sterols/100 g,with the majority of the sterols being free sterols.

As used herein, the term “seedoil” refers to a composition obtained fromthe seed/grain of a plant which comprises at least 60% (w/w) lipid, orobtainable from the seed/grain if the seedoil is still present in theseed/grain. That is, seedoil of the invention includes seedoil which ispresent in the seed/grain or portion thereof, as well as seedoil whichhas been extracted from the seed/grain. The seedoil is preferablyextracted seedoil. Seedoil is typically a liquid at room temperature.Preferably, the total fatty acid (TFA) content in the seedoilpredominantly (>50%) comprises fatty acids that are at least 16 carbonsin length. More preferably, at least 50% of the total fatty acids in theseedoil are C18 fatty acids for example, oleic acid. The fatty acids aretypically in an esterified form such as for example, TAG, DAG, acyl-CoAor phospholipid. The fatty acids may be free fatty acids and/or in anesterified form. In an embodiment, at least 50%, more preferably atleast 70%, more preferably at least 80%, more preferably at least 90%,more preferably at least 91%, more preferably at least 92%, morepreferably at least 93%, more preferably at least 94%, more preferablyat least 95%, more preferably at least 96%, more preferably at least97%, more preferably at least 98%, more preferably at least 99% of thefatty acids in seedoil of the invention can be found as TAG. In anembodiment, seedoil of the invention is “substantially purified” or“purified” oil that has been separated from one or more other lipids,nucleic acids, polypeptides, or other contaminating molecules with whichit is associated in the seed or in a crude extract. It is preferred thatthe substantially purified seedoil is at least 60% free, more preferablyat least 75% free, and more preferably, at least 90% free from othercomponents with which it is associated in the seed or extract. Seedoilof the invention may further comprise non-fatty acid molecules such as,but not limited to, sterols. In an embodiment, the seedoil is canola oil(Brassica sp. such as Brassica carinata, Brassica juncea, Brassicanapobrassica, Brassica napus) mustard oil (Brassica juncea), otherBrassica oil (e.g., Brassica napobrassica, Brassica camelina), sunfloweroil (Helianthus sp. such as Helianthus annuus), linseed oil (Linumusitalissimum), soybean oil (Glycine max), safflower oil (Carthamustinctorius), corn oil (Zea mays), tobacco oil (Nicotiana sp. such asNicotiana tabacum or Nicotiana benthamiana), peanut oil (Arachishypogaea), palm oil (Elaeis guineensis), cottonseed oil (Gossypiumhirsutum), coconut oil (Cocos nucifera), avocado oil (Persea americana),olive oil (Olea europaea), cashew oil (Anacardium occidentale),macadamia oil (Macadamia intergrifolia), almond oil (Prunus amygdalus),oat seed oil (Avena sativa), rice oil (Oryza sp. such as Oryza sativaand Oryza glaberrima), Arabidopsis seed oil (Arabidopsis thaliana), oroil from the seed of Acrocomia aculeata (macauba palm), Aracinishypogaea (peanut), Astrocaryum murumuru (murumuru), Astrocaryum vulgare(tucumã), Attalea geraensis (Indaiá-rateiro), Attalea humilis (Americanoil palm), Attalea oleifera (andaiá), Attalea phalerata (uricuri),Attalea speciosa (babassu), Beta vulgaris (sugar beet), Camelina sativa(false flax), Caryocar brasiliense (pequi), Crambe abyssinica(Abyssinian kale), Cucumis melo (melon), Hordeum vulgare (barley),Jatropha curcas (physic nut), Joannesia princeps (arara nut-tree),Licania rigida (oiticica), Lupinus angustifolius (lupin), Mauritiaflexuosa (buriti palm), Maximiliana maripa (inaja palm), Miscanthus sp.such as Miscanthus x giganteus and Miscanthus sinensis, Oenocarpusbacaba (bacaba-do-azeite), Oenocarpus bataua (pataul), Oenocarpusdistichus (bacaba-de-leque), Panicum virgatum (switchgrass), Paraqueibaparaensis (mari), Persea amencana (avocado), Pongamia pinnata (Indianbeech), Populus trichocarpa, Ricinus communis (castor), Saccharum sp.(sugarcane), Sesamum indicum (sesame), Solanum tuberosum (potato),Sorghum sp. such as Sorghum bicolor, Sorghum vulgare, Theobromagrandiforum (cupuassu), Trifolium sp., Trithrinax brasiliensis(Brazilian needle palm) and Triticum sp. (wheat) such as Triticumaestivum. Seedoil may be extracted from seed/grain by any method knownin the art. This typically involves extraction with nonpolar solventssuch as diethyl ether, petroleum ether, chloroform/methanol or butanolmixtures, generally associated with first crushing of the seeds. Lipidsassociated with the starch in the grain may be extracted withwater-saturated butanol. The seedoil may be “de-gummed” by methods knownin the art to remove polysaccharides or treated in other ways to removecontaminants or improve purity, stability, or colour. The TAGs and otheresters in the seedoil may be hydrolysed to release free fatty acids, orthe seedoil hydrogenated, treated chemically, or enzymatically as knownin the art.

As used herein, the term “fatty acid” refers to a carboxylic acid withan aliphatic tail of at least 8 carbon atoms in length, either saturatedor unsaturated. Preferred fatty acids have a carbon-carbon bonded chainof at least 12 carbons in length. Most naturally occurring fatty acidshave an even number of carbon atoms because their biosynthesis involvesacetate which has two carbon atoms. The fatty acids may be in a freestate (non-esterified) or in an esterified form such as part of a TAG,DAG. MAG, acyl-CoA (thio-ester) bound, acyl-ACP bound, or othercovalently bound form. When covalently bound in an esterified form, thefatty acid is referred to herein as an “acyl” group. The fatty acid maybe esterified as a phospholipid such as a phosphatidylcholine (PC),phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol,phosphatidylinositol, or diphosphatidylglycerol. Saturated fatty acidsdo not contain any double bonds or other functional groups along thechain. The term “saturated” refers to hydrogen, in that all carbons(apart from the carboxylic acid [—COOH] group) contain as many hydrogensas possible. In other words, the omega (ω) end contains 3 hydrogens(CH3-) and each carbon within the chain contains 2 hydrogens (—CH2-).Unsaturated fatty acids are of similar form to saturated fatty acids,except that one or more alkene functional groups exist along the chain,with each alkene substituting a singly-bonded “—CH2-CH2-” part of thechain with a doubly-bonded “—CH═CH—” portion (that is, a carbon doublebonded to another carbon). The two next carbon atoms in the chain thatare bound to either side of the double bond can occur in a cis or transconfiguration.

As used herein, the terms “monounsaturated fatty acid” or “MUFA” referto a fatty acid which comprises at least 12 carbon atoms in its carbonchain and only one alkene group (carbon-carbon double bond), which maybe in an esterified or non-esterified (free) form. As used herein, theterms “polyunsaturated fatty acid” or “PUFA” refer to a fatty acid whichcomprises at least 12 carbon atoms in its carbon chain and at least twoalkene groups (carbon-carbon double bonds), which may be in anesterified or non-esterified form.

As used herein, a fatty acid with a “medium chain length”, also referredto as “MCFA”, comprises an acyl chain of 8 to 14 carbons. The acyl chainmay be modified (for example it may comprise one or more double bonds, ahydroxyl group, an expoxy group, etc) or unmodified (saturated). Thisterms at least includes one or more or all of caprylic acid (C8:0),capric acid (C10:0), lauric acid (C12:0), and myristic acid (C14:0).

“Monoacylglyceride” or “MAG” is glyceride in which the glycerol isesterified with one fatty acid. As used herein, MAG comprises a hydroxylgroup at an sn-1/3 (also referred to herein as sn-1 MAG or 1-MAG or1/3-MAG) or sn-2 position (also referred to herein as 2-MAG), andtherefore MAG does not include phosphorylated molecules such as PA orPC. MAG is thus a component of neutral lipids in a cell.

“Diacylglyceride” or “DAG” is glyceride in which the glycerol isesterified with two fatty acids which may be the same or, preferably,different. As used herein, DAG comprises a hydroxyl group at a sn-1,3 orsn-2 position, and therefore DAG does not include phosphorylatedmolecules such as PA or PC. DAG is thus a component of neutral lipids ina cell. In the Kennedy pathway of DAG synthesis (FIG. 1), the precursorsn-glycerol-3-phosphate (G3P) is esterified to two acyl groups, eachcoming from a fatty acid coenzyme A ester, in a first reaction catalysedby a glycerol-3-phosphate acyltransferase (GPAT) at position sn-1 toform LysoPA, followed by a second acylation at position sn-2 catalysedby a lysophosphatidic acid acyltransferase (LPAAT) to form phosphatidicacid (PA). This intermediate is then de-phosphorylated by PAP to formDAG. DAG may also be formed from TAG by removal of an acyl group by alipase, or from PC essentially by removal of a choline headgroup by anyof the enzymes PDCT, PLC or PLD (FIG. 1).

“Triacylglyceride” or “TAG” is glyceride in which the glycerol isesterified with three fatty acids which may be the same (e.g. as intri-olein) or, more commonly, different. In the Kennedy pathway of TAGsynthesis, DAG is formed as described above, and then a third acyl groupis esterified to the glycerol backbone by the activity of DGAT.Alternative pathways for formation of TAG include one catalysed by theenzyme PDAT (FIG. 1) and the MGAT pathway described herein.

As used herein, the term “wild-type” or variations thereof refers to avegetative plant part, cell, seed or non-human organism or part thereof,such as a tuber or beet, that has not been genetically modified, such ascomprise an exogenous polynucleotyide(s), according to this invention.

The term “corresponding” refers to a vegetative plant part, a cell, seedor non-human organism or part thereof (such as a tuber or beet) that hasthe same or similar genetic background as a vegetative plant part, acell, seed or non-human organism or part thereof of the invention butwhich has not been modified as described herein (for example, avegetative plant part, a cell, seed or non-human organism or partthereof which lacks the exogenous polynucleotide(s) and/or lacks thegenetic modification(s)). In a preferred embodiment, the correspondingvegetative plant part, eukaryotic cell, seed or non-human organism orpart thereof is at the same developmental stage as the vegetative plantpart, eukaryotic cell, seed or non-human organism or part thereof of theinvention. For example, if the non-human organism is a flowering plant,then preferably the corresponding plant is also flowering. Acorresponding vegetative plant part, eukaryotic cell, seed or non-humanorganism or part thereof, can be used as a control to compare levels ofnucleic acid or protein expression, or the extent and nature of traitmodification, for example non-polar lipid production and/or content,with the vegetative plant part, eukaryotic cell, seed or non-humanorganism or part thereof of the invention which is modified as describedherein. A person skilled in the art is readily able to determine anappropriate “corresponding” vegetative plant part, eukaryotic cell, seedor non-human organism or part thereof, tissue, organ or organism forsuch a comparison.

As used herein, “compared with” or “relative to” refers to comparinglevels of a non-polar lipid, total non-polar lipid content, fatty acidcontent or other parameter of the vegetative plant part, eukaryoticcell, seed, non-human organism or part thereof (such as a tuber or beet)expressing the one or more exogenous polynucleotides or exogenouspolypeptides with a vegetative plant part, eukaryotic cell, seed,non-human organism or part thereof lacking the one or more exogenouspolynucelotides or polypeptides.

As used herein, “enhanced ability to produce non-polar lipid” is arelative term which refers to the total amount of non-polar lipid beingproduced by a vegetative plant part, eukaryotic cell, seed or non-humanorganism or part thereof (such as a tuber or beet) of the inventionbeing increased relative to a corresponding vegetative plant part,eukaryotic cell, seed or non-human organism or part thereof. In oneembodiment, the TAG and/or polyunsaturated fatty acid content, or theoleic acid content in the total fatty acid content of the non-polarlipid is increased, or the linolenic acid content in the total fattyacid content of the non-polar lipid is decreased, for example by atleast 2% in absolute terms.

As used herein, “synergism”, “synergistic”, “acting synergistically” andrelated terms are each a comparative term that means that the effect ofa combination of elements present in a cell, plant or part thereof ofthe invention, for example a combination of elements A and B, is greaterthan the sum of the effects of the elements separately in correspondingcells, plants or parts thereof, for example the sum of the effect of Aand the effect of B. Where more than two elements are present in thecell, plant or part thereof, for example elements A, B and C, it meansthat the effect of the combination of all of the elements is greaterthan the sum of the effects of the individual effects of the elements.In a preferred embodiment, it means that the effect of the combinationof elements A, B and C is greater than the sum of the effect of elementsA and B combined and the effect of element C. In such a case, it can besaid that element C acts synergistically with elements A and B. As wouldbe understood, the effects are measured in corresponding cells, plantsor parts thereof, for example grown under the same conditions and at thesame stage of biological development.

As used herein, “germinate at a rate substantially the same as for acorresponding wild-type plant” refers to seed of a plant of theinvention being relatively able to germinate when compared to seed of awild-type plant lacking the defined exogenous polynucleotide(s).Germination may be measured in vitro on tissue culture medium or in soilas occurs in the field. In one embodiment, the number of seeds whichgerminate, for instance when grown under optimal greenhouse conditionsfor the plant species, is at least 75%, more preferably at least 90%,when compared to corresponding wild-type seed. In another embodiment,the seeds which germinate, for instance when grown under optimalglasshouse conditions for the plant species, produce seedlings whichgrow at a rate which, on average, is at least 75%, more preferably atleast 90%, when compared to corresponding wild-type plants. This isreferred to as “seedling vigour”. In an embodiment, the rate of initialroot growth and shoot growth of seedlings of the invention isessentially the same compared to a corresponding wild-type seedlinggrown under the same conditions. In an embodiment, the leaf biomass (dryweight) of the plants of the invention is at least 80%, preferably atleast 90%, of the leaf biomass relative to a corresponding wild-typeplant grown under the same conditions, preferably in the field. In anembodiment, the height of the plants of the invention is at least 70%,preferably at least 80%, more preferably at least 90%, of the plantheight relative to a corresponding wild-type plant grown under the sameconditions, preferably in the field and preferably at maturity.

As used herein, the term “an exogenous polynucleotide whichdown-regulates the production and/or activity of an endogenouspolypeptide” or variations thereof, refers to a polynucleotide thatencodes an RNA molecule (for example, encoding an amiRNA or hpRNAi) thatdown-regulates the production and/or activity, or itself down-regulatesthe production and/or activity (for example, is an amiRNA or hpRNA whichcan be delivered directly to, for example, a cell) of an endogenouspolypeptide for example, SDP1 TAG lipase, plastidial GPAT, plastidialLPAAT, TGD polypeptide, AGPase, or delta-12 fatty acid desturase (FAD2),or a combination of two or more thereof. Typically, the RNA moleculedecreases the expression of an endogenous gene encoding the polypeptide.

As used herein, the term “on a weight basis” refers to the weight of asubstance (for example, TAG, DAG, fatty acid) as a percentage of theweight of the composition comprising the substance (for example, seed,leaf). For example, if a transgenic seed has 25 μg total fatty acid per120 μg seed weight; the percentage of total fatty acid on a weight basisis 20.8%.

As used herein, the term “on a relative basis” refers to a parametersuch as the amount of a substance in a composition comprising thesubstance in comparison with the parameter for a correspondingcomposition, as a percentage. For example, a reduction from 3 units to 2units is a reduction of 33% on a relative basis.

As used herein, “plastids” are organelles in plants, including algae,which are the site of manufacture of carbon-based compounds fromphotosynthesis including sugars, starch and fatty acids. Plastidsinclude chloroplasts which contain chlorophyll and carry outphotosynthesis, etioplasts which are the predecessors of chloroplasts,as well as specialised plastids such as chromoplasts which are colouredplastids for synthesis and storage of pigments, gerontoplasts whichcontrol the dismantling of the photosynthetic apparatus duringsenescence, amyloplasts for starch synthesis and storage, elaioplastsfor storage of lipids, and proteinoplasts for storing and modifyingproteins.

As used herein, the term “biofuel” refers to any type of fuel, typicallyas used to power machinery such as automobiles, planes, boats, trucks orpetroleum powered motors, whose energy is derived from biological carbonfixation. Biofuels include fuels derived from biomass conversion, aswell as solid biomass, liquid fuels and biogases. Examples of biofuelsinclude bioalcohols, biodiesel, synthetic diesel, vegetable oil,bioethers, biogas, syngas, solid biofuels, algae-derived fuel,biohydrogen, biomethanol, 2,5-Dimethylfuran (DMF), biodimethyl ether(bioDME), Fischer-Tropsch diesel, biohydrogen diesel, mixed alcohols andwood diesel.

As used herein, the term “bioalcohol” refers to biologically producedalcohols, for example, ethanol, propanol and butanol. Bioalcohols areproduced by the action of microorganisms and/or enzymes through thefermentation of sugars, hemicellulose or cellulose.

As used herein, the term “biodiesel” refers to a composition comprisingfatty acid methyl- or ethyl-esters derived from lipids bytransesterification, the lipids being from living cells not fossilfuels.

As used herein, the term “synthetic diesel” refers to a form of dieselfuel which is derived from renewable feedstock rather than the fossilfeedstock used in most diesel fuels.

As used herein, the term “vegetable oil” includes a pure plant oil (orstraight vegetable oil) or a waste vegetable oil (by product of otherindustries), including oil produced in either a vegetative plant part orin seed.

As used herein, the term “biogas” refers to methane or a flammablemixture of methane and other gases produced by anaerobic digestion oforganic material by anaerobes.

As used herein, the term “syngas” refers to a gas mixture that containsvarying amounts of carbon monoxide and hydrogen and possibly otherhydrocarbons, produced by partial combustion of biomass. Syngas may beconverted into methanol in the presence of catalyst (usuallycopper-based), with subsequent methanol dehydration in the presence of adifferent catalyst (for example, silica-alumina).

As used herein, the term “Fischer-Tropsch” refers to a set of chemicalreactions that convert a mixture of carbon monoxide and hydrogen intoliquid hydrocarbons. The syngas can first be conditioned using forexample, a water gas shift to achieve the required H₂/CO ratio. Theconversion takes place in the presence of a catalyst, usually iron orcobalt. The temperature, pressure and catalyst determine whether a lightor heavy syncrude is produced. For example at 330° C. mostly gasolineand olefins are produced whereas at 180° to 250° C. mostly diesel andwaxes are produced. The liquids produced from the syngas, which comprisevarious hydrocarbon fractions, are very clean (sulphur free)straight-chain hydrocarbons.

As used herein, the term “biochar” refers to charcoal made from biomass,for example, by pyrolysis of the biomass.

As used herein, the term “feedstock” refers to a material, for example,biomass or a conversion product thereof (for example, syngas) when usedto produce a product, for example, a biofuel such as biodiesel or asynthetic diesel.

As used herein, the term “industrial product” refers to a hydrocarbonproduct which is predominantly made of carbon and hydrogen such as fattyacid methyl- and/or ethyl-esters or alkanes such as methane, mixtures oflonger chain alkanes which are typically liquids at ambienttemperatures, a biofuel, carbon monoxide and/or hydrogen, or abioalcohol such as ethanol, propanol, or butanol, or biochar. The term“industrial product” is intended to include intermediary products thatcan be converted to other industrial products, for example, syngas isitself considered to be an industrial product which can be used tosynthesize a hydrocarbon product which is also considered to be anindustrial product. The term industrial product as used herein includesboth pure forms of the above compounds, or more commonly a mixture ofvarious compounds and components, for example the hydrocarbon productmay contain a range of carbon chain lengths, as well understood in theart.

As used herein, “progeny” means the immediate and all subsequentgenerations of offspring produced from a parent, for example a second,third or later generation offspring.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

The term “and/or”, e.g., “X and/or Y” shall be understood to mean either“X and Y” or “X or Y” and shall be taken to provide explicit support forboth meanings or for either meaning.

As used herein, the term about, unless stated to the contrary, refers to+/−10%, more preferably +/−5%, more preferably +/−2%, more preferably+/−1%, even more preferably +/−0.5%, of the designated value.

Production of Non-Polar Lipids and Triacylglycerols

The present invention is based on the finding that the non-polar lipidcontent in recombinant eukaryotic cells can be increased by acombination of modifications selected from those designated herein as:(A). Push, (B). Pull, (C). Protect, (D). Package, (E). PlastidialExport, (F). Plastidial Import and (G). Prokaryotic Pathway. Asdescribed herein, cells without plastids can comprise variouscombinations of A-D, whereas cells with plastids, such as plant andalgal cells, can comprise various combinations of A-G.

Recombinant cells, transgenic non-human animals or a part thereof, andtransgenic plants or part thereof, of the invention therefore have havea number of combinations of exogenous polynucleotides and/or geneticmodifications each of which provide for one of the modifications. Theseexogenous polynucleotides and/or genetic modifications include:

(A) an exogenous polynucleotide which encodes a transcription factorpolypeptide that increases the expression of one or more glycolyticand/or fatty acid biosynthetic genes in the cell, transgenic non-humananimal or a part thereof, or transgenic plant or part thereof, providingthe “Push” modification,

(B) an exogenous polynucleotide which encodes a polypeptide involved inthe biosynthesis of one or more non-polar lipids in the cell, transgenicnon-human animal or a part thereof, or transgenic plant or part thereof,providing the “Pull” modification,

(C) a genetic modification which down-regulates endogenous productionand/or activity of a polypeptide involved in the catabolism oftriacylglycerols (TAG) in the cell, transgenic non-human animal or apart thereof, or transgenic plant or part thereof when compared to acorresponding the cell, transgenic non-human animal or a part thereof,or transgenic plant or part thereof lacking the genetic modification,providing the “Protect” modification,

(D) an exogenous polynucleotide which encodes an oil body coating (OBC)polypeptide, providing the “Package” modification,

(E) an exogenous polynucleotide which encodes a polypeptide whichincreases the export of fatty acids out of plastids of the cell,transgenic non-human animal or a part thereof, or transgenic plant orpart thereof, when compared to a corresponding cell, transgenicnon-human animal or a part thereof, or transgenic plant or part thereoflacking the exogenous polynucleotide, providing the “Plastidial Export”modification,

(F) a genetic modification which down-regulates endogenous productionand/or activity of a polypeptide involved in importing fatty acids intoplastids of the cell, transgenic non-human animal or a part thereof, ortransgenic plant or part thereof when compared to a corresponding cell,transgenic non-human animal or a part thereof, or transgenic plant orpart thereof lacking the genetic modification, providing the “PlastidialImport” modification, and

G) a genetic modification which down-regulates endogenous productionand/or activity of a polypeptide involved in diacylglycerol (DAG)production in the plastid of the cell, transgenic non-human animal or apart thereof, or transgenic plant or part thereof when compared to acorresponding cell, transgenic non-human animal or a part thereof, ortransgenic plant or part thereof lacking the genetic modification,providing the “prokaryotic Pathway” modification.

Preferred combinations (also referred to herein as sets) of exogenouspolynucleotides and/or genetic modifications of the invention are;

1) A, B and optionally one of C, D, E, F or G;

2) A, C and optionally one of D, E, F or G;

3) A, D and optionally one of E, F or G;

4) A, E and optionally F or G;

5) A, F and optionally G;

6) A and G;

7) A, B, C and optionally one of D, E, F or G;

8) A, B, D and optionally one of E, F or G;

9) A, B, E and optionally F or G;

10) A, B, F and optionally G;

11) A, B, C, D and optionally one of E, F or G;

12) A, B, C, E and optionally F or G;

13) A, B, C, F and optionally G;

14) A, B, D, E and optionally F or G;

15) A, B, D, F and optionally G;

16) A, B, E, F and optionally G;

17) A, C. D and optionally one of E, F or G;

18) A, C, E and optionally F or G;

19) A, C, F and optionally G;

20) A, C, D, E and optionally F or G;

21) A, C, D, F and optionally G;

22) A, C, E, F and optionally a fifth modification G;

23) A, D, E and optionally F or G;

24) A, D, F and optionally G;

25) A, D, E, F and optionally G;

26) A, E, F and optionally G;

27) Six of A, B, C, D, E, F and G omitting one of A, B, C, D, E, F or G,and

28) Any one of 1-26 above where there are two or more exogenouspolynucleotides encoding two or more different transcription factorpolypeptides that increases the expression of one or more glycolyticand/or fatty acid biosynthetic genes in the cell, for example oneexogenous polynucleotide encoding WRI1 and another exogenouspolynucleotide encoding LEC2.

In each of the above preferred combinations there may be at least twodifferent exogenous polynucleotides which encode at least two differenttranscription factor polypeptides that increases the expression of oneor more glycolytic and/or fatty acid biosynthetic genes in the cell,transgenic non-human animal or a part thereof, or transgenic plant orpart thereof.

These modifications are described as follows:

A. The “Push” modification is characterised by an increased synthesis oftotal fatty acids in the plastids of the eukaryotic cell. In anembodiment, this occurs by the increased expression and/or activity of atranscription factor which regulates fatty acid synthesis in theplastids. In one embodiment, this can be achieved by expressing in atransgenic cell an exogenous polynucleotide which encodes atranscription factor polypeptide that increases the expression of one ormore glycolytic and/or fatty acid biosynthetic genes in the cell. In anembodiment, the increased fatty acid synthesis is not caused by theprovision to the cell of an altered ACCase whose activity is lessinhibited by fatty acids, relative to the endogenous ACCase in the cell.In an embodiment, the cell comprises an exogenous polynucleotide whichencodes the transcription factor, preferably under the control of apromoter other than a constitutive promoter. The transcription factormay be selected from the group consisting of WRI1, LEC1, LEC1-like,LEC2, BBM, FUS3, ABI3, ABI4, ABI5, Dof4, Dof11 or the group consistingof MYB73, bZIP53, AGL15, MYB115, MYB118, TANMEI, WUS, GFR2a1, GFR2a2 andPHR1, and is preferably WRI1, LEC1 or LEC2. In a further embodiment, theincreased synthesis of total fatty acids is relative to a correspondingwild-type cell. In an embodiment, there are two or more exogenouspolynucleotides encoding two or more different transcription factorpolypeptides.

B. The “Pull” modification is characterised by increased expressionand/or activity in the cell of a fatty acyl acyltransferase whichcatalyses the synthesis of TAG, DAG or MAG in the cell, such as a DGAT,PDAT, LPAAT, GPAT or MGAT, preferably a DGAT or a PDAT. In oneembodiment, this can be achieved by expressing in a transgenic cell anexogenous polynucleotide which encodes a polypeptide involved in thebiosynthesis of one or more non-polar lipids. In an embodiment, theacyltransferase is a membrane-bound acyltransferase that uses anacyl-CoA substrate as the acyl donor in the case of DGAT, LPAAT, GPAT orMGAT, or an acyl group from PC as the acyl donor in the case of PDAT.The Pull modification can be relative to a corresponding wild-type cellor, preferably, relative to a corresponding cell which has the Pushmodification. In an embodiment, the cell comprises an exogenouspolynucleotide which encodes the fatty acyl acyltransferase.

C. The “Protect” modification is characterised by a reduction in thecatabolism of triacylglycerols (TAG) in the cell. In an embodiment, thiscan be achieved through a genetic modification in the cell whichdown-regulates endogenous production and/or activity of a polypeptideinvolved in the catabolism of triacylglycerols (TAG) in the cell whencompared to a corresponding cell lacking the genetic modification. Inembodiment, the cell has a reduced expression and/or activity of anendogenous TAG lipase in the cell, preferably an SDP1 lipase, a Cgi58polypeptide, an acyl-CoA oxidase such as the ACX1 or ACX2, or apolypeptide involved in β-oxidation of fatty acids in the cell such as aPXA1 peroxisomal ATP-binding cassette transporter. This may occur byexpression in the cell of an exogenous polynucleotide which encodes anRNA molecule which reduces the expression of, for example, an endogenousgene encoding the TAG lipase such as the SDP1 lipase, acyl-CoA oxidaseor the polypeptide involved in β-oxidation of fatty acids in the cell,or by a mutation in an endogenous gene encoding, for example, the TAGlipase, acyl-CoA oxidase or polypeptide involved in β-oxidation of fattyacids. In an embodiment, the reduced expression and/or activity isrelative to a corresponding wild-type cell or relative to acorresponding cell which has the Push modification.

D. The “Package” modification is characterised by an increasedexpression and/or accumulation of an oil body coating (OBC) polypeptide.In an embodiment, this can be achieved by expressing in a transgeniccell an exogenous polynucleotide which encodes an oil body coating (OBC)polypeptide. The OBC polypeptide may be an oleosin, such as for examplea polyoleosin, a caoleosin or a steroleosin, or preferably an LDAP. Inan embodiment, the level of oleosin that is accumulated in theeukaryotic cell is at least 2-fold higher relative to the correspondingcell comprising the oleosin gene from the T-DNA of pJP3502. In anembodiment, the increased expression or accumulation of the OBCpolypeptide is not caused solely by the Push modification. In anembodiment, the expression and/or accumulation is relative to acorresponding wild-type cell or, preferably, relative to a correspondingcell which has the Push modification.

E. The “Plastidial Export” modification is characterised by an increasedrate of export of total fatty acids out of the plastids of theeukaryotic cell. In one embodiment, this can be achieved by expressingin a transgenic cell an exogenous polynucleotide which encodes apolypeptide which increases the export of fatty acids out of plastids ofthe cell when compared to a corresponding cell lacking the exogenouspolynucleotide. In an embodiment, this occurs by the increasedexpression and/or activity of a fatty acid thioesterase (TE), a fattyacid transporter polypeptide such as an ABCA9 polypeptide, or along-chain acyl-CoA synthetase (LACS). In an embodiment, the cellcomprises an exogenous polynucleotide which encodes the TE, fatty acidtransporter polypeptide or LACS. The TE may be a FATB polypeptide orpreferably a FATA polypeptide. In an embodiment, the TE is preferably aTE with specificity for MCFA. In an embodiment, the Plastidial Exportmodification is relative to a corresponding wild-type cell or,preferably, relative to a corresponding cell which has the Pushmodification.

F. The “Plastidial Import” modification is characterised by a reducedrate of import of fatty acids into the plastids of the cell from outsideof the plastids. In an embodiment, this can be achieved through agenetic modification in the cell which down-regulates endogenousproduction and/or activity of a polypeptide involved in importing fattyacids into plastids of the cell when compared to a corresponding celllacking the genetic modification. For example, this may occur byexpression in the cell of an exogenous polynucleotide which encodes anRNA molecule which reduces the expression of an endogenous gene encodingan transporter polypeptide such as a TGD polypeptide, for example aTGD1, TGD2, TGD3 or TGD4 polypeptide, or by a mutation in an endogenousgene encoding the TGD polypeptide. In an embodiment, the reduced rate ofimport is relative to a corresponding wild-type cell or relative to acorresponding cell which has the Push modification.

G. The “Prokaryotic Pathway” modification is characterised by adecreased amount of DAG or rate of production of DAG in the plastids ofthe cell. In an embodiment, this can be achieved through a geneticmodification in the cell which down-regulates endogenous productionand/or activity of a polypeptide involved in diacylglycerol (DAG)production in the plastid when compared to a corresponding cell lackingthe genetic modification. In an embodiment, the decreased amount or rateof production of DAG occurs by a decreased production of LPA fromacyl-ACP and G3P in the plastids. The decreased amount or rate ofproduction of DAG may occur by expression in the cell of an exogenouspolynucleotide which encodes an RNA molecule which reduces theexpression of an endogenous gene encoding a plastidial GPAT, plastidialLPAAT or a plastidial PAP, preferably a plastidial GPAT, or by amutation in an endogenous gene encoding the plastidial polypeptide. Inan embodiment, the decreased amount or rate of production of DAG isrelative to a corresponding wild-type cell or, preferably, relative to acorresponding cell which has the Push modification.

The Push modification is essential to the invention, and the Pullmodification is preferred. The Protect and Package modifications may becomplementary i.e. one of the two may be sufficient. The cell maycomprise one, two or all three of the Plastidial Export, PlastidialImport and Prokaryotic Pathway modifications. In an embodiment, at leastone of the exogenous polynucleotides in the cell, preferably at leastthe exogenous polynucleotide encoding the transcription factor whichregulates fatty acid synthesis in the plastids, is expressed under thecontrol of (H) a promoter other than a constitutive promoter such as,for example, a developmentally related promoter, a promoter that ispreferentially active in photosynthetic cells, a tissue-specificpromoter, a promoter which has been modified by reducing its expressionlevel relative to a corresponding native promoter, or is preferably asenesence-specific promoter. More preferably, at least the exogenouspolynucleotide encoding the transcription factor which regulates fattyacid synthesis in the plastids is expressed under the control of apromoter other than a constitutive promoter and the exogenouspolynucleotide which encodes an RNA molecule which down-regulatesendogenous production and/or activity of a polypeptide involved in thecatabolism of triacylglycerols is also expressed under the control of apromoter other than a constitutive promoter, which promoters may be thesame or different.

Plants produce some, but not all, of their membrane lipids such as MGDGin plastids by the so-called prokaryotic pathway (FIG. 1). In plants,there is also a eukaryotic pathway for synthesis of galactolipids andglycerolipids which synthesizes FA first of all in the plastid and thenassembles the FA into glycerolipids in the ER. MGDG synthesised by theeukaryotic pathway contains C18:3 (ALA) fatty acid esterified at thesn-2 position of MGDG. The DAG backbone including the ALA for the MGDGsynthesis by this pathway is assembled in the ER and then imported intothe plastid. In contrast, the MGDG synthesized by the prokaryoticpathway contains C16:3 fatty acid esterified at the sn-2 position ofMGDG. The ratio of the contribution of the prokaryotic pathway relativeto the eukaryotic pathway in producing MGDG (16:3) vs MGDG (18:3) is acharacteristic and distinctive feature of different plant species(Mongrand et al. 1998). This distinctive fatty acid composition of MGDGallows all higher plants (angiosperms) to be classified as eitherso-called 16:3 or 18:3 plants. 16:3 species, exemplified by Arabidopsisand Brassica napus, generally have both of the prokaryotic andeukaryotic pathways of MGDG synthesis operating, whereas the 18:3species exemplified by Nicotiana tabacum, Pisum sativum and Glycine maxgenerally have only (or almost entirely) the eukaryotic pathway of MGDGsynthesis, providing little or no C16:3 fatty acid accumulation in thevegetative tissues. As used herein, a “16:3 plant” or “16:3 species” isone which has more than 2% C16:3 fatty acid in the total fatty acidcontent of its photosynthetic tissues. As used herein, a “18:3 plant” or“18:3 species” is one which has less than 2% C16:3 fatty acid in thetotal fatty acid content of its photosynthetic tissues. As describedherein, a plant can be converted from being a 16:3 plant to an 18:3plant by suitable genetic modifications. The proportion of flux betweenthe prokaryote and eukaryote pathways is not conserved across differentplant species or tissues. In 16:3 species up to 40% of flux in leavesoccurs via the prokaryotic pathway (Browse et al., 1986), while in 18:3species, such as pea and soybean, about 90% of FAs which are synthesizedin the plastid are exported out of the plastid to the ER to supply thesource of FA for the eukaryotic pathway (Ohlrogge and Browse, 1995;Somerville et al., 2000).

Therefore different amounts of 18:3 and 16:3 fatty acids are foundwithin the glycolipids of different plant species. This is used todistinguish between 18:3 plants whose fatty acids with 3 double bondsare almost entirely C18 fatty acids and the 16:3 plants that containboth C₁₆- and C₁₈-fatty acids having 3 double bonds. In chloroplasts of18:3 plants, enzymic activities catalyzing the conversion ofphosphatidate to diacylglycerol and of diacylglycerol to monogalactosyldiacylglycerol (MGD) are significantly less active than in 16:3chloroplasts. In leaves of 18:3 plants, chloroplasts synthesizestearoyl-ACP2 in the stroma, introduce the first double bond into thesaturated hydrocarbon chain, and then hydrolyze the thioester bythioesterases (FIG. 1). Released oleate is exported across chloroplastenvelopes into membranes of the eucaryotic part of the cell, probablythe endoplasmic reticulum, where it is incorporated into PC. PC-linkedoleoyl groups are desaturated in these membranes and subsequently moveback into the chloroplast. The MGD-linked acyl groups are substrates forthe introduction of the third double bond to yield MGD with twolinolenoyl residues. This galactolipid is characteristic of 18:3 plantssuch as Asteraceae and Fabaceae, for example. In photosyntheticallyactive cells of 16:3 plants which are represented, for example, bymembers of Apiaceae and Brassicaceae, two pathways operate in parallelto provide thylakoids with MGD.

In one embodiment, the vegetative plant part, eukaryotic cell, seed ortransgenic non-human organism or part thereof (such as a tuber or beet)of the invention produces higher levels of non-polar lipids such as TAG,or total fatty acid (TFA) content, preferably both, than a correspondingvegetative plant part, eukaryotic cell, seed or non-human organism orpart thereof which lacks the genetic modifications or exogenouspolynucleotides. In one example, plants of the invention produce seeds,leaves, or have leaf portions of at least 1 cm² in surface area, stemsand/or tubers having an increased non-polar lipid content such as TAG orTFA content, preferably both, when compared to corresponding seeds,leaves, leaf portions of at least 1 cm² in surface area, stems ortubers.

In another embodiment, the vegetative plant part, transgenic non-humanorganism or part thereof (such as a tuber or beet), preferably a plant,tuber, beet or seed, produce TAGs that are enriched for one or moreparticular fatty acids. A wide spectrum of fatty acids can beincorporated into TAGs, including saturated and unsaturated fatty acidsand short-chain and long-chain fatty acids. Some non-limiting examplesof fatty acids that can be incorporated into TAGs and which may beincreased in level include: capric (10:0), lauric (12:0), myristic(14:0), palmitic (16:0), palmitoleic (16:1), stearic (18:0), oleic(18:1), vaccenic (18:1), linoleic (18:2), eleostearic (18:3),γ-linolenic (18:3), α-linolenic (18:3ω3), stearidonic (18:4ω3),arachidic (20:0), eicosadienoic (20:2), dihomo-γ-linoleic (20:3),eicosatrienoic (20:3), arachidonic (20:4), eicosatetraenoic (20:4),eicosapentaenoic (20:5ω3), behenic (22:0), docosapentaenoic (22:5ω),docosahexaenoic (22:6ω3), lignoceric (24:0), nervonic (24:1), cerotic(26:0), and montanic (28:0) fatty acids. In one embodiment of thepresent invention, the vegetative plant part, eukaryotic cell, seed ortransgenic organism or parts thereof (such as a tuber or beet) isenriched for TAGs comprising oleic acid, and/or is reduced in linolenicacid (ALA), preferably by at least 2% or at least 5% on an absolutebasis.

Preferably, the vegetative plant part, eukaryotic cell, seed ortransgenic non-human organism or part thereof of the invention aretransformed with one or more chimeric DNAs (exogenous polynucleotides).In the case of multiple chimeric DNAs, these are preferably covalentlylinked on one DNA molecule such as, for example, a single T-DNAmolecule, and preferably integrated at a single locus in the host cellgenome. Alternatively, the chimeric DNAs are on two or more DNAmolecules which may be unlinked in the host genome, or the DNAmolecule(s) is not integrated into the host genome, such as occurs intransient expression experiments. The plant, vegetative plant part,eukaryotic cell, seed or transgenic non-human organism or part thereofis preferably homozygous for the one DNA molecule inserted into itsgenome.

Transcription Factors

Various transcription factors are involved in eukaryotic cells in thesynthesis of fatty acids and lipids incorporating the fatty acids suchas TAG, and therefore can be manipulated for the Push modification. Apreferred transcription factor is WRI1. As used herein, the term“Wrinkled 1” or “WRI1” or “WRL1” refers to a transcription factor of theAP2/ERWEBP class which regulates the expression of several enzymesinvolved in glycolysis and de novo fatty acid biosynthesis. WRI1 has twoplant-specific (AP2/EREB) DNA-binding domains. WRI1 in at leastArabidopsis also regulates the breakdown of sucrose via glycolysisthereby regulating the supply of precursors for fatty acid biosynthesis.In other words, it controls the carbon flow from the photosynthate tostorage lipids. wri1 mutants in at least Arabidopsis have a wrinkledseed phenotype, due to a defect in the incorporation of sucrose andglucose into TAGs.

Examples of genes which are transcribed by WRI1 include, but are notlimited to, one or more, preferably all, of genes encoding pyruvatekinase (At5 g52920, At3 g22960), pyruvate dehydrogenase (PDH) E1alphasubunit (At1g01090), acetyl-CoA carboxylase (ACCase), BCCP2 subunit(At5g15530), enoyl-ACP reductase (At2 g05990; EAR), phosphoglyceratemutase (At1g22170), cytosolic fructokinase, and cytosolicphosphoglycerate mutase, sucrose synthase (SuSy) (see, for example, Liuet al., 2010b; Baud et al., 2007; Ruuska et al., 2002).

WRI1 contains the conserved domain AP2 (cd00018). AP2 is a DNA-bindingdomain found in transcription regulators in plants such as APETALA2 andEREBP (ethylene responsive element binding protein). In EREBPs thedomain specifically binds to the 11 bp GCC box of the ethylene responseelement (ERE), a promotor element essential for ethylene responsiveness.EREBPs and the C-repeat binding factor CBF1, which is involved in stressresponse, contain a single copy of the AP2 domain. APETALA2-likeproteins, which play a role in plant development contain two copies.

Other sequence motifs which may be found in WRI1 and its functionalhomologs include:

1. (SEQ ID NO: 89) R G V T/S R H R W T G R. 2. (SEQ ID NO: 90)F/Y E A H L W D K. 3. (SEQ ID NO: 91) D L A A L K Y W G. 4.(SEQ ID NO: 92) S X G F S/A R G X. 5. (SEQ ID NO: 93)H H H/Q N G R/K W E A R I G R/K V. 6. (SEQ ID NO: 94) Q E E A A A X Y D.

As used herein, the term “Wrinkled 1” or “WRI1” also includes “Wrinkled1-like” or “WRI1-like” proteins. Examples of WRI1 proteins includeAccession Nos: Q6X5Y6, (Arabidopsis thaliana; SEQ ID NO:22),XP_002876251.1 (Arabidopsis lyrata subsp. Lyrata; SEQ ID NO:23),ABD16282.1 (Brassica napus; SEQ ID NO:24), ADO16346.1 (Brassica napus;SEQ ID NO:25), XP_003530370.1 (Glycine max; SEQ ID NO:26), AEO22131.1(Jatropha curcas; SEQ ID NO:27), XP_002525305.1 (Ricinus communis; SEQID NO:28), XP_002316459.1 (Populus trichocarpa; SEQ ID NO:29),CBI29147.3 (Vitis vinifera; SEQ ID NO:30), XP_003578997.1 (Brachypodiumdistachyon; SEQ ID NO:31), BAJ86627.1 (Hordeum vulgare subsp. vulgare;SEQ ID NO:32), EAY79792.1 (Oryza sativa; SEQ ID NO:33), XP_002450194.1(Sorghum bicolor; SEQ ID NO:34), ACG32367.1 (Zea mays; SEQ ID NO:35),XP_003561189.1 (Brachypodium distachyon; SEQ ID NO:36), ABL85061.1(Brachypodium sylvaticum; SEQ ID NO:37), BAD68417.1 (Oryza sativa; SEQID NO:38), XP_002437819.1 (Sorghum bicolor; SEQ ID NO:39),XP_002441444.1 (Sorghum bicolor; SEQ ID NO:40), XP_003530686.1 (Glycinemax; SEQ ID NO:41), XP_003553203.1 (Glycine max; SEQ ID NO:42),XP_002315794.1 (Populus trichocarpa; SEQ ID NO:43), XP_002270149.1(Vitis vinifera; SEQ ID NO:44), XP_003533548.1 (Glycine max; SEQ IDNO:45), XP_003551723.1 (Glycine max; SEQ ID NO:46), XP_003621117.1(Medicago truncatula; SEQ ID NO:47), XP_002323836.1 (Populustrichocarpa; SEQ ID NO:48), XP_002517474.1 (Ricinus communis; SEQ IDNO:49), CAN79925.1 (Vitis vinifera; SEQ ID NO:50), XP_003572236.1(Brachypodium distachyon; SEQ ID NO:51), BAD10030.1 (Oryza sativa; SEQID NO:52), XP_002444429.1 (Sorghum bicolor; SEQ ID NO:53),NP_001170359.1 (Zea mays; SEQ ID NO:54), XP_002889265.1 (Arabidopsislyrata subsp. lyrata; SEQ ID NO:55), AAF68121.1 (Arabidopsis thaliana;SEQ ID NO:56), NP_178088.2 (Arabidopsis thaliana; SEQ ID NO:57),XP_002890145.1 (Arabidopsis lyrata subsp. lyrata; SEQ ID NO:58),BAJ33872.1 (Thellungiella halophila; SEQ ID NO:59), NP_563990.1(Arabidopsis thaliana; SEQ ID NO:60), XP_003530350.1 (Glycine max; SEQID NO:61), XP_003578142.1 (Brachypodium distachyon; SEQ ID NO:62),EAZ09147.1 (Oryza sativa; SEQ ID NO:63), XP_002460236.1 (Sorghumbicolor; SEQ ID NO:64), NP_001146338.1 (Zea mays; SEQ ID NO:65),XP_003519167.1 (Glycine max; SEQ ID NO:66), XP_003550676.1 (Glycine max;SEQ ID NO:67), XP_003610261.1 (Medicago truncatula; SEQ ID NO:68),XP_003524030.1 (Glycine max; SEQ ID NO:69), XP_003525949.1 (Glycine max;SEQ ID NO:70), XP_002325111.1 (Populus trichocarpa; SEQ ID NO:71),CB136586.3 (Vitis vinifera; SEQ ID NO:72), XP_002273046.2 (Vitisvinifera; SEQ ID NO:73), XP_002303866.1 (Populus trichocarpa; SEQ IDNO:74), and CB125261.3 (Vitis vinifera; SEQ ID NO:75). Further examplesinclude Sorbi-WRL1 (SEQ ID NO:76), Lupan-WRL1 (SEQ ID NO:77), Ricco-WRL1(SEQ ID NO:78), and Lupin angustifolius WRI1 (SEQ ID NO:79). A preferredWRI1 is a maize WRI1 or a sorghum WRI1.

More recently, a subset of WRI1-like transcription factors have beenre-classified as WRI2, WRI3 or WRI4 transcription factors, which arecharacterised by preferential expression in stems and/or roots of plantsrather than in developing seeds (To et al., 2012). Despite theirre-classification, these are included in the definition of “WRI1”herein. Preferred WRI1-like transcription factors are those which cancomplement the function of a wri1 mutation in a plant, particularly thefunction in developing seed of the plant such as in an A. thaliana wri1mutant. The function of a WRI1-like polypeptide can also be assayed inthe N. benthamiana transient assays as described herein.

As used herein, a “LEAFY COTYLEDON” or “LEC” polypeptide means atranscription factor which is a LEC1, LEC1-like, LEC2, ABI3 or FUS3transcription factor which exhibits broad control on seed maturation andfatty acid synthesis. LEC2, FUS3 and ABI3 are related polypeptides thateach contain a B3 DNA-binding domain of 120 amino acids (Yamasaki etal., 2004) that is only found in plant proteins. They can bedistinguished by phylogenetic analysis to determine relatedness in aminoacid sequence to the members of the A. thaliana polypeptides having theAccession Nos as follows: LEC2, Accession No. AAL12004.1; FUS3 (alsoknown as FUSCA3), Accession No. AAC35247. LEC1 belongs to adifferent-class of polypeptides and is homologous to a HAP3 polypeptideof the CBF binding factor class (Lee et al., 2003). The LEC1, LEC2 andFUS3 genes are required in early embryogenesis to maintain embryoniccell fate and to specify cotyledon identity and in later in initiationand maintenance of embryo maturation (Santos-Mendoza et al., 2008). Theyalso induce expression of genes encoding seed storage proteins bybinding to RY motifs present in the promoters, and oleosin genes. Theycan also be distinguished by their expression patterns in seeddevelopment or by their ability to complement the corresponding mutationin A. thaliana.

As used herein, the term “Leafy Cotyledon 1” or “LEC1” refers to aNF-YB-type transcription factor which participates in zygoticdevelopment and in somatic embryogenesis. The endogenous gene isexpressed specifically in seed in both the embryo and endosperm. LEC1activates the gene encoding WRI1 as well as a large class of fatty acidsynthesis genes. Ectopic expression of LEC2 also causes rapid activationof auxin-responsive genes and may cause formation of somatic embryos.Examples of LEC1 polypeptides include proteins from Arabidopsis thaliana(AAC39488, SEQ ID NO:149), Medicago truncatula (AFK49653, SEQ ID NO:154)and Brassica napus (ADF81045, SEQ ID NO:151), A. lyrata (XP_002862657,SEQ ID NO:150), R. communis (XP_002522740, SEQ ID NO:152), G. max(XP_006582823, SEQ ID NO:153), A. hypogaea (ADC33213, SEQ ID NO:156), Z.mays (AAK95562, SEQ ID NO:155).

LEC1-like (L1L) is closely related to LEC1 but has a different patternof gene expression, being expressed earlier during embryogenesis (Kwonget al., 2003). Examples of LEC1-like polypeptides include proteins fromArabidopsis thaliana (AAN15924, SEQ ID NO:157), Brassica napus(AH194922, SEQ ID NO:158), and Phaseolus coccineus LEC1-like (AAN01148,SEQ ID NO: 159).

As used herein, the term “Leafy Cotyledon 2” or “LEC2” refers to a B3domain transcription factor which participates in zygotic developmentand in somatic embryogenesis and which activates expression of a geneencoding WRI1. Its ectopic expression facilitates the embryogenesis fromvegetative plant tissues (Alemanno et al., 2008). Examples of LEC2polypeptides include proteins from Arabidopsis thaliana (Accession No.NP_564304.1, SEQ ID NO:142), Medicago truncatula (Accession No.CAA42938.1, SEQ ID NO:143) and Brassica napus (Accession No. ADO16343.1,SEQ ID NO:144).

In an embodiment, an exogenous polynucleotide of the invention whichencodes a LEC2 comprises one or more of the following:

i) nucleotides encoding a polypeptide comprising amino acids whosesequence is set forth as any one of SEQ ID NOs:142 to 144, or abiologically active fragment thereof, or a polypeptide whose amino acidsequence is at least 30% identical to any one or more of SEQ ID NOs:142to 144,

ii) nucleotides whose sequence is at least 30% identical to i), and

iii) a polynucleotide which hybridizes to one or both of i) or ii) understringent conditions.

As used herein, the term “FUS3” refers to a B3 domain transcriptionfactor which participates in zygotic development and in somaticembryogenesis and is detected mainly in the protodermal tissue of theembryo (Gazzarrini et al., 2004). Examples of FUS3 polypeptides includeproteins from Arabidopsis thaliana (AAC35247, SEQ ID NO:160), Brassicanapus (XP_006293066.1, SEQ ID NO:161) and Medicago truncatula(XP_003624470, SEQ ID NO: 162). Over-expression of any of LEC1, L1L,LEC2, FUS3 and ABI3 from an exogenous polynucleotide is preferablycontrolled by a developmentally regulated promoter such as a senescencespecific promoter, an inducible promoter, or a promoter which has beenengineered for providing a reduced level of expression relative to anative promoter, particularly in plants other than Arabidopsis thalianaand B. napus cv. Westar, in order to avoid developmental abnormalitiesin plant development that are commonly associated with over-expressionof these transcription factors (Mu et al., 2008).

As used herein, the term “BABY BOOM” or “BBM” refers an AP2/ERFtranscription factor that induces regeneration under culture conditionsthat normally do not support regeneration in wild-type plants. Ectopicexpression of Brassica napus BBM (BnBBM) genes in B. napus andArabidopsis induces spontaneous somatic embryogenesis and organogenesisfrom seedlings grown on hormone-free basal medium (Boutilier et al.,2002). In tobacco, ectopic BBM expression is sufficient to induceadventitious shoot and root regeneration on basal medium, but exogenouscytokinin is required for somatic embryo (SE) formation (Srinivasan etal., 2007). Examples of BBM polypeptides include proteins fromArabidopsis thaliana (Accession No. NP_197245.2, SEQ ID NO:145), maize(U.S. Pat. No. 7,579,529), Sorghum bicolor (Accession No. XP_002458927)and Medicago truncatula (Accession No. AAW82334.1, SEQ ID NO:146).

In an embodiment, an exogenous polynucleotide of the invention whichencodes BBM comprises, unless specified otherwise, one or more of thefollowing:

i) nucleotides encoding a polypeptide comprising amino acids whosesequence is set forth as one of SEQ ID NOs:145 or 146, or a biologicallyactive fragment thereof, or a polypeptide whose amino acid sequence isat least 30% identical to one or both of SEQ ID NOs: 145 or 146,

ii) nucleotides whose sequence is at least 30% identical to i), and

iii) a polynucleotide which hybridizes to one or both of i) or ii) understringent conditions.

An ABI3 polypeptide (A. thaliana Accession No. NP_189108) is related tothe maize VP1 protein, is expressed at low levels in vegetative tissuesand affects plastid development. An ABI4 polypeptide (A. thalianaAccession NP_181551) belongs to a family of transcription factors thatcontain a plant-specific AP2 domain (Finkelstein et al., 1998) and actsdownstream of ABI3. ABI5 (A. thaliana Accession No. NP_565840) is atranscription factor of the bZIP family which affects ABA sensitivityand controls the expression of some LEA genes in seeds. It binds to anABA-responsive element.

Each of the following transcription factors was selected on the basisthat they functioned in embryogenesis in plants. Accession numbers areprovided in Table 10. Homologs of each can be readily identified in manyother plant species and tested as described in Example 10.

MYB73 is a transcription factor that has been identified in soybean,involved in stress responses.

bZIP53 is a transcription factor in the bZIP protein family, identifiedin Arabidopsis.

AGL15 (Agamous-like 15) is a MADS box transcription factor which isnatively expressed during embryogenesis. AGL15 is also nativelyexpressed in leaf primordia, shoot apical meristems and young floralbuds, suggesting that AGL15 may also have a function duringpost-germinative development. AGL15 has a role in embryogenesis andgibberellic acid catabolism. It targets B3 domain transcription factorsthat are key regulators of embryogenesis.

MYB115 and MYB118 are transcription factors in the MYB family fromArabidopsis involved in embryogenesis.

TANMEI also known as EMB2757 encodes a WD repeat protein required forembryo development in Arabidopsis.

WUS, also known as Wuschel, is a homeobox gene that controls the stemcell pool in embryos. It is expressed in the stem cell organizing centerof meristems and is required to keep the stem cells in anundifferentiated state. The transcription factor binds to a TAAT elementcore motif.

GFR2a1 and GFR2a2 are transcription factors at least from soybean.

Fatty Acyl Acyltransferases

As used herein, the term “fatty acyl acyltransferase” refers to aprotein which is capable of transferring an acyl group from acyl-CoA, PCor acyl-ACP, preferably acyl-CoA or PC, onto a substrate to form TAG,DAG or MAG. These acyltransferases include DGAT, PDAT, MGAT, GPAT andLPAAT.

As used herein, the term “diacylglycerol acyltransferase” (DGAT) refersto a protein which transfers a fatty acyl group from acyl-CoA to a DAGsubstrate to produce TAG. Thus, the term “diacylglycerol acyltransferaseactivity” refers to the transfer of an acyl group from acyl-CoA to DAGto produce TAG. A DGAT may also have MGAT function but predominantlyfunctions as a DGAT, i.e., it has greater catalytic activity as a DGATthan as a MGAT when the enzyme activity is expressed in units of nmolesproduct/min/mg protein (see for example, Yen et al., 2005). The activityof DGAT may be rate-limiting in TAG synthesis in seeds (Ichihara et al.,1988). DGAT uses an acyl-CoA substrate as the acyl donor and transfersit to the sn-3 position of DAG to form TAG. The enzyme functions in itsnative state in the endoplasmic reticulum (ER) of the cell.

There are three known types of DGAT, referred to as DGAT1, DGAT2 andDGAT3, respectively. DGAT1 polypeptides are membrane proteins thattypically have 10 transmembrane domains, DGAT2 polypeptides are alsomembrane proteins but typically have 2 transmembrane domains, whilstDGAT3 polypeptides typically have none and are thought to be soluble inthe cytoplasm, not integrated into membranes. Plant DGAT1 polypeptidestypically have about 510-550 amino acid residues while DGAT2polypeptides typically have about 310-330 residues. DGAT1 is the mainenzyme responsible for producing TAG from DAG in most developing plantseeds, whereas DGAT2s from plant species such as tung tree (Verniciafordii) and castor bean (Ricinus communis) that produce high amounts ofunusual fatty acids appear to have important roles in the accumulationof the unusual fatty acids in TAG. Over-expression of AtDGAT1 in tobaccoleaves resulted in a 6-7 fold increased TAG content (Bouvier-Nave etal., 2000).

Examples of DGAT1 polypeptides include DGAT1 proteins from Aspergillusfumigatus (XP_755172.1; SEQ ID NO:80), Arabidopsis thaliana (CAB44774.1;SEQ ID NO:1), Ricinus communis (AAR11479.1; SEQ ID NO:81), Verniciafordii (ABC94472.1; SEQ ID NO:82), Vernonia galamensis (ABV21945.1 andABV21946.1; SEQ ID NO:83 and SEQ ID NO:84, respectively), Euonymusalatus (AAV31083.1; SEQ ID NO:85), Caenorhabditis elegans (AAF82410.1;SEQ ID NO:86), Rattus norvegicus (NP_445889.1; SEQ ID NO:87), Homosapiens (NP_036211.2; SEQ ID NO:88), as well as variants and/or mutantsthereof. Examples of DGAT2 polypeptides include proteins encoded byDGAT2 genes from Arabidopsis thaliana (NP_566952.1; SEQ ID NO:2),Ricinus communis (AAY16324.1; SEQ ID NO:3), Vernicia fordii (ABC94474.1;SEQ ID NO:4), Mortierella ramanniana (AAK84179.1; SEQ ID NO:5), Homosapiens (Q96PD7.2; SEQ ID NO:6) (Q58HT5.1; SEQ ID NO:7), Bos taurus(Q70VZ8.1; SEQ ID NO:8), Mus musculus (AAK84175.1; SEQ ID NO:9), as wellas variants and/or mutants thereof. DGAT1 and DGAT2 amino acid sequencesshow little homology. Expression in leaves of an exogenous DGAT2 wastwice as effective as a DGAT1 in increasing oil content (TAG). Further,A. thaliana DGAT2 had a greater preference for linoleoyl-CoA andlinolenoyl-CoA as acyl donors relative to oleoyl-CoA, compared to DGAT1.This substrate preference can be used to distinguish the two DGATclasses in addition to their amino acid sequences.

Examples of DGAT3 polypeptides include proteins encoded by DGAT3 genesfrom peanut (Arachis hypogaea, Saha, et al., 2006), as well as variantsand/or mutants thereof. A DGAT has little or no detectable MGATactivity, for example, less than 300 pmol/min/mg protein, preferablyless than 200 pmol/min/mg protein, more preferably less than 100pmol/min/mg protein.

In an embodiment, an exogenous polynucleotide of the invention whichencodes a DGAT1 comprises one or more of the following:

i) nucleotides encoding a polypeptide comprising amino acids whosesequence is set forth as any one of SEQ ID NOs:1 or 80 to 88, or abiologically active fragment thereof, or a polypeptide whose amino acidsequence is at least 30% identical to any one or more of SEQ ID NOs: 1or 80 to 88,

ii) nucleotides whose sequence is at least 30% identical to i), and

iii) a polynucleotide which hybridizes to one or both of i) or ii) understringent conditions.

In an embodiment, an exogenous polynucleotide of the invention whichencodes a DGAT2 comprises one or more of the following:

i) nucleotides encoding a polypeptide comprising amino acids whosesequence is set forth as any one of SEQ ID NOs:2 to 9, or a biologicallyactive fragment thereof, or a polypeptide whose amino acid sequence isat least 30% identical to any one or more of SEQ ID NOs: 2 to 9,

ii) nucleotides whose sequence is at least 30% identical to i), and

iii) a polynucleotide which hybridizes to one or both of i) or ii) understringent conditions.

As used herein, the term “phospholipid:diacylglycerol acyltransferase”(PDAT; EC 2.3.1.158) or its synonym “phospholipid:1,2-diacyl-sn-glycerolO-acyltransferase” means an acyltransferase that transfers an acyl groupfrom a phospholipid, typically PC, to the sn-3 position of DAG to formTAG. This reaction is unrelated to DGAT and uses phospholipids as theacyl-donors. There are several forms of PDAT in plant cells includingPDAT1, PDAT2 or PDAT3 (Ghosal et al., 2007).

As used herein, the term “monoacylglycerol acyltransferase” or “MGAT”refers to a protein which transfers a fatty acyl group from acyl-CoA toa MAG substrate, for example sn-2 MAG, to produce DAG. Thus, the term“monoacylglycerol acyltransferase activity” at least refers to thetransfer of an acyl group from acyl-CoA to MAG to produce DAG. The term“MGAT” as used herein includes enzymes that act on sn-1/3 MAG and/orsn-2 MAG substrates to form sn-1,3 DAG and/or sn-1,2/2,3-DAG,respectively. In a preferred embodiment, the MGAT has a preference forsn-2 MAG substrate relative to sn-1 MAG, or substantially uses only sn-2MAG as substrate. As used herein, MGAT does not include enzymes whichtransfer an acyl group preferentially to LysoPA relative to MAG, suchenzymes are known as LPAATs. That is, a MGAT preferentially usesnon-phosphorylated monoacyl substrates, even though they may also havelow catalytic activity on LysoPA. A preferred MGAT does not havedetectable activity in acylating LysoPA. A MGAT may also have DGATfunction but predominantly functions as a MGAT, i.e., it has greatercatalytic activity as a MGAT than as a DGAT when the enzyme activity isexpressed in units of nmoles product/min/mg protein (also see Yen etal., 2002). There are three known classes of MGAT, referred to as,MGAT1, MGAT2 and MGAT3, respectively. Examples of MGAT1, MGAT2 and MGAT3polypeptides are described in WO2013/096993.

As used herein, an “MGAT pathway” refers to an anabolic pathway,different to the Kennedy pathway for the formation of TAG, in which DAGis formed by the acylation of either sn-1 MAG or preferably sn-2 MAG,catalysed by MGAT. The DAG may subsequently be used to form TAG or otherlipids. WO2012/000026 demonstrated firstly that plant leaf tissue cansynthesise MAG from G-3-P such that the MAG is accessible to anexogenous MGAT expressed in the leaf tissue, secondly MGAT from varioussources can function in plant tissues, requiring a successfulinteraction with other plant factors involved in lipid synthesis andthirdly the DAG produced by the exogenous MGAT activity is accessible toa plant DGAT, or an exogenous DGAT, to produce TAG. MGAT and DGATactivity can be assayed by introducing constructs encoding the enzymes(or candidate enzymes) into Saccharomyces cerevisiae strain H1246 anddemonstrating TAG accumulation.

Some of the motifs that have been shown to be important for catalyticactivity in some DGAT2s are also conserved in MGAT acyltransferases. Ofparticular interest is a putative neutral lipid-binding domain with theconcensus sequence FLXLXXXN (SEQ ID NO:14) where each X is independentlyany amino acid other than proline, and N is any nonpolar amino acid,located within the N-terminal transmembrane region followed by aputative glycerol/phospholipid acyltransferase domain. The FLXLXXXNmotif (SEQ ID NO:14) is found in the mouse DGAT2 (amino acids 81-88) andMGAT1/2 but not in yeast or plant DGAT2s. It is important for activityof the mouse DGAT2. Other DGAT2 and/or MGAT1/2 sequence motifs include:

1. A highly conserved YFP tripeptide (SEQ ID NO:10) in most DGAT2polypeptides and also in MGAT1 and MGAT2, for example, present as aminoacids 139-141 in mouse DGAT2. Mutating this motif within the yeast DGAT2with non-conservative substitutions rendered the enzyme non-functional.2. HPHG tetrapeptide (SEQ ID NO: 11), highly conserved in MGATs as wellas in DGAT2 sequences from animals and fungi, for example, present asamino acids 161-164 in mouse DGAT2, and important for catalytic activityat least in yeast and mouse DGAT2. Plant DGAT2 acyltransferases have aEPHS (SEQ ID NO:12) conserved sequence instead, so conservative changesto the first and fourth amino acids can be tolerated.3. A longer conserved motif which is part of the putative glycerolphospholipid domain. An example of this motif isRXGFX(K/R)XAXXXGXXX(L/V)VPXXXFG(E/Q) (SEQ ID NO:13), which is present asamino acids 304-327 in mouse DGAT2. This motif is less conserved inamino acid sequence than the others, as would be expected from itslength, but homologs can be recognised by motif searching. The spacingmay vary between the more conserved amino acids, i.e., there may beadditional X amino acids within the motif, or less X amino acidscompared to the sequence above.

One important component in glycerolipid synthesis from fatty acidsesterified to ACP or CoA is the enzyme sn-glycerol-3-phosphateacyltransferase (GPAT), which is another of the polypeptides involved inthe biosynthesis of non-polar lipids. This enzyme is involved indifferent metabolic pathways and physiological functions. It catalysesthe following reaction: G3P+fatty acyl-ACP or -CoA→LPA+free-ACP or -CoA.The GPAT-catalyzed reaction occurs in three distinct plant subcellularcompartments: plastid, endoplasmic reticulum (ER) and mitochondria.These reactions are catalyzed by three different types of GPAT enzymes,a soluble form localized in plastidial stroma which uses acyl-ACP as itsnatural acyl substrate (PGPAT in FIG. 1), and two membrane-bound formslocalized in the ER and mitochondria which use acyl-CoA and acyl-ACP asnatural acyl donors, respectively (Chen et al., 2011).

As used herein, the term “glycerol-3-phosphate acyltransferase” (GPAT;EC 2.3.1.15) and its synonym “glycerol-3-phosphate O-acyltransferase”refer to a protein which acylates glycerol-3-phosphate (G-3-P) to formLysoPA and/or MAG, the latter product forming if the GPAT also hasphosphatase activity on LysoPA. The acyl group that is transferred isfrom acyl-CoA if the GPAT is an ER-type GPAT (an“acyl-CoA:sn-glycerol-3-phosphate 1-O-acyltransferase” also referred toas “microsomal GPAT”) or from acyl-ACP if the GPAT is a plastidial-typeGPAT (PGPAT). Thus, the term “glycerol-3-phosphate acyltransferaseactivity” refers to the acylation of G-3-P to form LysoPA and/or MAG.The term “GPAT” encompasses enzymes that acylate G-3-P to form sn-1 LPAand/or sn-2 LPA, preferably sn-2 LPA. Preferably, the GPAT which may beover-expressed in the Pull modification is a membrane bound GPAT thatfunctions in the ER of the cell, more preferably a GPAT9, and theplastidial GPAT that is down-regulated in the Prokaryotic Pathwaymodification is a soluble GPAT (“plastidial GPAT”). In a preferredembodiment, the GPAT has phosphatase activity. In a most preferredembodiment, the GPAT is a sn-2 GPAT having phosphatase activity whichproduces sn-2 MAG.

As used herein, the term “sn-1 glycerol-3-phosphate acyltransferase”(sn-1 GPAT) refers to a protein which acylates sn-glycerol-3-phosphate(G-3-P) to preferentially form 1-acyl-sn-glycerol-3-phosphate (sn-1LPA). Thus, the term “sn-1 glycerol-3-phosphate acyltransferaseactivity” refers to the acylation of sn-glycerol-3-phosphate to form1-acyl-sn-glycerol-3-phosphate (sn-1 LPA).

As used herein, the term “sn-2 glycerol-3-phosphate acyltransferase”(sn-2 GPAT) refers to a protein which acylates sn-glycerol-3-phosphate(G-3-P) to preferentially form 2-acyl-sn-glycerol-3-phosphate (sn-2LPA). Thus, the term “sn-2 glycerol-3-phosphate acyltransferaseactivity” refers to the acylation of sn-glycerol-3-phosphate to form2-acyl-sn-glycerol-3-phosphate (sn-2 LPA).

The GPAT family is large and all known members contain two conserveddomains, a plsC acyltransferase domain (PF01553; SEQ ID NO:15) and aHAD-like hydrolase (PF12710; SEQ ID NO:16) superfamily domain andvariants thereof. In addition to this, at least in Arabidopsis thaliana,GPATs in the subclasses GPAT4-GPAT8 all contain a N-terminal regionhomologous to a phosphoserine phosphatase domain (PF00702; SEQ IDNO:17), and GPATs which produce MAG as a product can be identified bythe presence of such a homologous region. Some GPATs expressedendogenously in leaf tissue comprise the conserved amino acid sequenceGDLVICPEGITCREP (SEQ ID NO:18). GPAT4 and GPAT6 both contain conservedresidues that are known to be critical to phosphatase activity,specifically conserved amino acids in Motif I (DXDX[TN/V][L/V]; SEQ IDNO:19) and Motif III (K-[G/S][D/S]XXX[D/N]; SEQ ID NO:20) located at theN-terminus (Yang et al., 2010).

Homologues of Arabidopsis GPAT4 (Accession No. NP_171667.1) and GPAT6(NP_181346.1) include AAF02784.1 (Arabidopsis thaliana), AAL32544.1(Arabidopsis thaliana), AAP03413.1 (Oryza sativa), ABK25381.1 (Piceasitchensis), ACN34546.1 (Zea Mays), BAF00762.1 (Arabidopsis thaliana),BAH00933.1 (Oryza sativa), EAY84189.1 (Oryza sativa), EAY98245.1 (Oryzasativa), EAZ21484.1 (Oryza sativa), EEC71826.1 (Oryza sativa),EEC76137.1 (Oryza sativa), EEE59882.1 (Oryza sativa), EFJ08963.1(Selaginella moellendorffii), EFJ11200.1 (Selaginella moellendorfi),NP_001044839.1 (Oryza sativa), NP_001045668.1 (Oryza sativa),NP_001147442.1 (Zea mays), NP_001149307.1 (Zea mays), NP_001168351.1(Zea mays), AFH02724.1 (Brassica napus) NP_191950.2 (Arabidopsisthaliana), XP_001765001.1 (Physcomitrella patens), XP_001769671.1(Physcomitrella patens), (Vitis vinifera), XP_002275348.1 (Vitisvinifera), XP_002276032.1 (Vitis vinifera), XP_002279091.1 (Vitisvinifera), XP_002309124.1 (Populus trichocarpa), XP_002309276.1 (Populustrichocarpa), XP_002322752.1 (Populus trichocarpa), XP_002323563.1(Populus trichocarpa), XP_002439887.1 (Sorghum bicolor), XP_002458786.1(Sorghum bicolor), XP_002463916.1 (Sorghum bicolor), XP_002464630.1(Sorghum bicolor), XP_002511873.1 (Ricinus communis), XP_002517438.1(Ricinus communis), XP_002520171.1 (Ricinus communis), ACT32032.1(Vernicia fordii), NP_001051189.1 (Oryza sativa), AFH02725.1 (Brassicanapus), XP_002320138.1 (Populus trichocarpa), XP_002451377.1 (Sorghumbicolor), XP_002531350.1 (Ricinus communis), and XP_002889361.1(Arabidopsis lyrata).

The soluble plastidial GPATs (PGPAT, also known as ATS1 in Arabidopsisthaliana) have been purified and genes encoding them cloned from severalplant species such as pea (Pisum sativum, Accession number: P30706.1),spinach (Spinacia oleracea, Accession number: Q43869.1), squash(Cucurbita moschate, Accession number: P10349.1), cucumber (Cucumissativus, Accession number: Q39639.1) and Arabidopsis thaliana (Accessionnumber: Q43307.2). The soluble plastidial GPAT is the first committedstep for what is known as the prokaryotic pathway of glycerolipidsynthesis and is operative only in the plastid (FIG. 1). The so-calledprokaryotic pathway is located exclusively in plant plastids andassembles DAG for the synthesis of galactolipids (MGDG and DGMG) whichcontain C16:3 fatty acids esterified at the sn-2 position of theglycerol backbone.

Conserved motifs and/or residues can be used as a sequence-baseddiagnostic for the identification of GPAT enzymes. Alternatively, a morestringent function-based assay could be utilised. Such an assayinvolves, for example, feeding labelled glycerol-3-phosphate to cells ormicrosomes and quantifying the levels of labelled products by thin-layerchromatography or a similar technique. GPAT activity results in theproduction of labelled LPA whilst GPAT/phosphatase activity results inthe production of labelled MAG.

As used herein, the term “lysophosphatidic acid acyltransferase” (LPAAT;EC 2.3.1.51) and its synonyms “I-acyl-glycerol-3-phosphateacyltransferase”, “acyl-CoA:1-acyl-sn-glycerol-3-phosphate2-O-acyltransferase” and “1-acylglycerol-3-phosphate O-acyltransferase”refer to a protein which acylates lysophosphatidic acid (LPA) to formphosphatidic acid (PA). The acyl group that is transferred is fromacyl-CoA if the LPAAT is an ER-type LPAAT or from acyl-ACP if the LPAATis a plastidial-type LPAAT (PLPAAT). Thus, the term “lysophosphatidicacid acyltransferase activity” refers to the acylation of LPA to formPA.

Oil Body Coating Polypeptides

Plant seeds and pollen accumulate TAG in subcellular structures calledoil bodies which generally range from 0.5-2.5 μm in diameter. As usedherein, “lipid droplets”, also referred to as “oil bodies”, are lipidrich cellular organelles for storage or exchange of neutral lipidsincluding predominantly TAG. Lipid droplets can vary greatly in sizefrom about 20 nm to 100 μm. These organelles have a TAG core surround bya phospholipid monolayer containing several embedded proteins which areinvolved in lipid metabolism and storage as well as lipid trafficking toother membranes, including oleosins if the oil bodies are from plantseeds or floral tissues (Jolivet et al., 2004). They generally consistof 0.5-3.5% protein while the remainder is the lipid. They are the leastdense of the organelles in most cells and can therefore be isolatedreadily by flotation centrifugation. Oleosins represent the mostabundant (at least 80%) of the protein in the membrane of oil bodiesfrom seeds.

As used herein, the term “Oleosin” refers to an amphipathic proteinpresent in the membrane of oil bodies in the storage tissues of seeds(see, for example, Huang, 1996; Lin et al., 2005; Capuano et al., 2007;Lui et al., 2009; Shimada and Hara-Nishimura, 2010) and artificiallyproduced variants (see for example WO2011/053169 and WO2011/127118).

Oleosins are of low M (15-26,000), corresponding to about 140-230 aminoacid residues, which allows them to become tightly packed on the surfaceof oil bodies. Within each seed species, there are usually two or moreoleosins of different Mr. Each oleosin molecule contains a relativelyhydrophilic, variable N-terminal domain (for example, about 48 aminoacid residues), a central totally hydrophobic domain (for example, ofabout 70-80 amino acid residues) which is particularly rich in aliphaticamino acids such as alanine, glycine, leucine, isoleucine and valine,and an amphipathic α-helical domain of about 30-40 amino acid residuesat or near the C-terminus. The central hydrophobic domain typicallycontains a proline knot motif of about 12 residues at its center.Generally, the central stretch of hydrophobic residues is inserted intothe lipid core and the amphiphatic N-terminal and/or amphiphaticC-terminal are located at the surface of the oil bodies, with positivelycharged residues embedded in a phospholipid monolayer and the negativelycharged ones exposed to the exterior.

As used herein, the term “Oleosin” encompasses polyoleosins which havemultiple oleosin polypeptides fused together in a head-to-tail fashionas a single polypeptide (WO2007/045019), for example 2×, 4× or 6×oleosin peptides, and caleosins which bind calcium and which are a minorprotein component of the proteins that coat oil bodies in seeds(Froissard et al., 2009), and steroleosins which bind sterols(WO2011/053169). However, generally a large proportion (at least 80%) ofthe oleosins of oil bodies will not be caleosins and/or steroleosins.The term “oleosin” also encompasses oleosin polypeptides which have beenmodified artificially, such oleosins which have one or more amino acidresidues of the native oleosins artificially replaced with cysteineresidues, as described in WO2011/053169. Typically, 4-8 residues aresubstituted artificially, preferably 6 residues, but as many as between2 and 14 residues can be substituted. Preferably, both of theamphipathic N-terminal and C-terminal domains comprise cysteinesubstitutions. The modification increases the cross-linking ability ofthe oleosins and increases the thermal stability and/or the stability ofthe proteins against degradation by proteases.

A substantial number of oleosin protein sequences, and nucleotidesequences encoding therefor, are known from a large number of differentplant species. Examples include, but are not limited to, oleosins fromArabidposis, canola, corn, rice, peanut, castor, soybean, flax, grape,cabbage, cotton, sunflower, sorghum and barley. Examples of oleosins(with their Accession Nos) include Brassica napus oleosin (CAA57545.1;SEQ ID NO:95), Brassica napus oleosin S1-1 (ACG69504.1; SEQ ID NO:96),Brassica napus oleosin S2-1 (ACG69503.1; SEQ ID NO:97), Brassica napusoleosin S3-1 (ACG69513.1; SEQ ID NO:98), Brassica napus oleosin S4-1(ACG69507.1; SEQ ID NO:99), Brassica napus oleosin S5-1 (ACG69511.1; SEQID NO:100), Arachis hypogaea oleosin 1 (AAZ20276.1; SEQ ID NO:101),Arachis hypogaea oleosin 2 (AAU21500.1; SEQ ID NO:102), Arachis hypogaeaoleosin 3 (AAU21501.1; SEQ ID NO:103), Arachis hypogaea oleosin 5(ABC96763.1; SEQ ID NO:104), Ricinus communis oleosin 1 (EEF40948.1; SEQID NO:105), Ricinus communis oleosin 2 (EEF51616.1; SEQ ID NO:106),Glycine max oleosin isoform a (P29530.2; SEQ ID NO:107), Glycine maxoleosin isoform b (P29531.1; SEQ ID NO: 108), Linum usitatissimumoleosin low molecular weight isoform (ABB01622.1; SEQ ID NO:109), Linumusitatissimum oleosin high molecular weight isoform (ABB01624.1; SEQ IDNO: 110), Helianthus annuus oleosin (CAA44224.1; SEQ ID NO:111), Zeamays oleosin (NP_001105338.1; SEQ ID NO:112), Brassica napus steroleosin(ABM30178.1; SEQ ID NO:113), Brassica napus steroleosin SLO1-1(ACG69522.1; SEQ ID NO:114), Brassica napus steroleosin SLO2-1(ACG69525.1; SEQ ID NO:115), Sesamum indicum steroleosin (AAL13315.1;SEQ ID NO:116), Zea mays steroleosin (NP_001152614.1; SEQ ID NO:117),Brassica napus caleosin CLO-1 (ACG69529.1; SEQ ID NO:118), Brassicanapus caleosin CLO-3 (ACG69527.1; SEQ ID NO:119), Sesamum indicumcaleosin (AAF13743.1; SEQ ID NO:120), Zea mays caleosin (NP_001151906.1;SEQ ID NO:121), Glycine max caleosin (AAB71227). Other lipidencapsulation polypeptides that are functionally equivalent areplastoglobulins and MLDP polypeptides (WO2011/127118).

In an embodiment, an exogenous polynucleotide of the invention whichencodes an oleosin comprises, unless specified otherwise, one or more ofthe following:

i) nucleotides encoding a polypeptide comprising amino acids whosesequence is set forth as any one of SEQ ID NOs:95 to 112, or abiologically active fragment thereof, or a polypeptide whose amino acidsequence is at least 30% identical to any one or more of SEQ ID NOs: 95to 112,

ii) nucleotides whose sequence is at least 30% identical to i), and

iii) a polynucleotide which hybridizes to one or both of i) or ii) understringent conditions.

In an embodiment, an exogenous polynucleotide of the invention whichencodes an steroleosin comprises, unless specified otherwise, one ormore of the following:

i) nucleotides encoding a polypeptide comprising amino acids whosesequence is set forth as any one of SEQ ID NOs: 113 to 117, or abiologically active fragment thereof, or a polypeptide whose amino acidsequence is at least 30% identical to any one or more of SEQ ID NOs: 113to 117,

ii) nucleotides whose sequence is at least 30% identical to i), and

iii) a polynucleotide which hybridizes to one or both of i) or ii) understringent conditions.

As used herein, a “lipid droplet associated protein” or “LDAP” means apolypeptide which is associated with lipid droplets in plants in tissuesor organs other than seeds, anthers and pollen, such as fruit tissuesincluding pericarp and mesocarp. LDAPs may be associated with oil bodiesin seeds, anthers or pollen as well as in the tissues or organs otherthan seeds, anthers and pollen. They are distinct from oleosins whichare polypeptides associated with the surface of lipid droplets in seedtissues, anthers and pollen. LDAPs as used herein include LDAPpolypeptides that are produced naturally in plant tissues as well asamino acid sequence variants that are produced artificially. Thefunction of such variants can be tested as exemplified in Example 15.

Horn et al. (2013) identified two LDAP genes which are expressed inavocado pericarp. The encoded avocado LDAP1 and LDAP2 polypeptides were62% identical in amino acid sequence and had homology to polypeptideencoded by Arabidopsis At3 g05500 and a rubber tree SRPP-like protein.Gidda et al. (2013) identified three LDAP genes that were expressed inoil palm (Elaeis guineensis) mesocarp but not in kernels and concludedthat LDAP genes were plant specific and conserved amongst all plantspecies. LDAP polypeptides may contain additional domains (Gidda et al.,(2013). Genes encoding LDAPs are generally up-regulated in non-seedtissues with abundant lipid and can be identified thereby, but arethought to be expressed in all non-seed cells that produce oil includingfor transient storage. Horn et al. (2013) shows a phylogenetic tree ofSRPP-like proteins in plants. Exemplary LDAP polypeptides are describedin Example 15 herein. Homologs of LDAPs in other plant species can bereadily identified by those skilled in the art.

In an embodiment, an exogenous polynucleotide of the invention whichencodes a LDAP comprises, unless specified otherwise, one or more of thefollowing:

i) nucleotides encoding a polypeptide comprising amino acids whosesequence is set forth as any one of SEQ ID NOs: 237, 239 or 241, or abiologically active fragment thereof, or a polypeptide whose amino acidsequence is at least 30% identical to any one or more of SEQ ID NOs:237, 239 or 241,

ii) nucleotides whose sequence is at least 30% identical to i), and

iii) a polynucleotide which hybridizes to one or both of i) or ii) understringent conditions.

As used herein, the term a “polypeptide involved in starch biosynthesis”refers to any polypeptide, the downregulation of which in a cell belownormal (wild-type) levels results in a reduction in the level of starchsynthesis and a decrease in the levels of starch. An example of such apolypeptide is AGPase.

As used herein, the term “ADP-glucose phosphorylase” or “AGPase” refersto an enzyme which regulates starch biosynthesis, catalysing conversionof glucose-1-phosphate and ATP to ADP-glucose which serves as thebuilding block for starch polymers. The active form of the AGPase enzymeconsists of 2 large and 2 small subunits.

The ADPase enzyme in plants exists primarily as a tetramer whichconsists of 2 large and 2 small subunits. Although these subunits differin their catalytic and regulatory roles depending on the species (Kuhnet al., 2009), in plants the small subunit generally displays catalyticactivity. The molecular weight of the small subunit is approximately50-55 kDa. The molecular weight of the large large subunit isapproximately 55-60 kDa. The plant enzyme is strongly activated by3-phosphoglycerate (PGA), a product of carbon dioxide fixation; in theabsence of PGA, the enzyme exhibits only about 3% of its activity. PlantAGPase is also strongly inhibited by inorganic phosphate (Pi). Incontrast, bacterial and algal AGPase exist as homotetramers of 50 kDa.The algal enzyme, like its plant counterpart, is activated by PGA andinhibited by Pi, whereas the bacterial enzyme is activated byfructose-1, 6-bisphosphate (FBP) and inhibited by AMP and Pi.

TAG Lipases and Beta-Oxidation

As used herein, the term “polypeptide involved in the degradation oflipid and/or which reduces lipid content” refers to any polypeptidewhich catabolises lipid, the downregulation of which in a cell belownormal (wild-type) levels results an increase in the level of oil, suchas fatty acids and/or TAGs, in the cell, preferably a cell of avegetative part, tuber, beet or a seed of a plant. Examples of suchpolypeptides include, but are not limited to, lipases, or a lipase suchas a CGi58 (Comparative Gene identifier-58-Like) polypeptide, aSUGAR-DEPENDENT1 (SDP1) triacylglycerol lipase (see, for example, Kellyet al., 2011) and a lipase described in WO 2009/027335.

As used herein, the term “TAG lipase” (EC.3.1.1.3) refers to a proteinwhich hydrolyzes TAG into one or more fatty acids and any one of DAG,MAG or glycerol. Thus, the term “TAG lipase activity” refers to thehydrolysis of TAG into glycerol and fatty acids.

As used herein, the term “CGi58” refers to a soluble acyl-CoA-dependentlysophosphatidic acid acyltransferase encoded by the At4 g24160 gene inArabidopsis thaliana and its homologs in other plants and “Ict1p” inyeast and its homologs. The plant gene such as that from Arabidopsisgene locus At4 g24160 is expressed as two alternative transcripts: alonger full-length isoform (At4 g24160.1) and a smaller isoform (At4g24160.2) missing a portion of the 3′ end (see James et al., 2010; Ghoshet al., 2009; US 201000221400). Both mRNAs code for a protein that ishomologous to the human CGI-58 protein and other orthologous members ofthis α/β hydrolase family (ABHD). In an embodiment, the CGI58 (At4g24160) protein contains three motifs that are conserved across plantspecies: a GXSXG lipase motif (SEQ ID NO:127), a HX(4)D acyltransferasemotif (SEQ ID NO: 128), and VX(3)HGF, a probable lipid binding motif(SEQ ID NO:129). The human CGI-58 protein has lysophosphatidic acidacyltransferase (LPAAT) activity but not lipase activity. In contrast,the plant and yeast proteins possess a canonical lipase sequence motifGXSXG (SEQ ID NO:127), that is absent from vertebrate (humans, mice, andzebrafish) proteins, and have lipase and phospholipase activity (Ghoshet al., 2009). Although the plant and yeast CGI58 proteins appear topossess detectable amounts of TAG lipase and phospholipase A activitiesin addition to LPAAT activity, the human protein does not.

Disruption of the homologous CGI-58 gene in Arabidopsis thaliana resultsin the accumulation of neutral lipid droplets in mature leaves. Massspectroscopy of isolated lipid droplets from cgi-58 loss-of-functionmutants showed they contain triacylglycerols with common leaf-specificfatty acids. Leaves of mature cgi-58 plants exhibit a marked increase inabsolute triacylglycerol levels, more than 10-fold higher than inwild-type plants. Lipid levels in the oil-storing seeds of cgi-58loss-of-function plants were unchanged, and unlike mutations inβ-oxidation, the cgi-58 seeds germinated and grew normally, requiring norescue with sucrose (James et al., 2010).

Examples of nucleotides encoding CGi58 polypeptides include those fromArabidopsis thaliana (NM_118548.1 encoding NP_194147.2; SEQ ID NO:130),Brachypodium distachyon (XP_003578450.1; SEQ ID NO:131), Glycine max(XM_003523590.1 encoding XP_003523638.1; SEQ ID NO:132), Zea mays(NM_001155541.1 encoding NP_001149013.1; SEQ ID NO:133), Sorghum bicolor(XM_002460493.1 encoding XP_002460538.1; SEQ ID NO:134), Ricinuscommunis (XM_002510439.1 encoding XP_002510485.1; SEQ ID NO:135),Medicago truncatula (XM_003603685.1 encoding XP_003603733.1; SEQ IDNO:136), and Oryza sativa (encoding EAZ09782.1).

In an embodiment, a genetic modification of the invention down-regulatesendogenous production of CGi58, wherein CGi58 is encoded by one or moreof the following:

i) nucleotides comprising a sequence set forth as any one of SEQ IDNOs:130 to 136,

ii) nucleotides comprising a sequence which is at least 30% identical toany one or more of SEQ ID NOs: 130 to 136, and

iii) a polynucleotide which hybridizes to one or both of i) or ii) understringent conditions.

Other lipases which have lipase activity on TAG include SUGAR-DEPENDENT1triacylglycerol lipase (SDP1, see for example Eastmond et al., 2006;Kelly et al., 2011) and SDP1-like polypeptides found in plant species aswell as yeast (TGL4 polypeptide) and animal cells, which are involved instorage TAG breakdown. The SDP1 and SDP1-like polypeptides appear to beresponsible for initiating TAG breakdown in seeds following germination(Eastmond et al., 2006). Plants that are mutant in SDP1, in the absenceof exogenous WRI1 and DGAT1, exhibit increased levels of PUFA in theirTAG. As used herein, “SDP1 polypeptides” include SDP1 polypeptides,SDP1-like polypeptides and their homologs in plant species. SDP1 andSDP1-like polypeptides in plants are 800-910 amino acid residues inlength and have a patatin-like acylhydrolase domain that can associatewith oil body surfaces and hydrolyse TAG in preference to DAG or MAG.SDP1 is thought to have a preference for hydrolysing the acyl group atthe sn-2 position of TAG. Arabidopsis contains at least three genesencoding SDP1 lipases, namely SDP1 (Accession No. NP_196024, nucleotidesequence SEQ ID NO:163 and homologs in other species), SDP1L (AccessionNo. NM_202720 and homologs in other species, Kelly et al., 2011) andATGLL (At1g33270) (Eastmond et al, 2006). Of particular interest forreducing gene activity are SDP1 genes which are expressed in vegetativetissues in plants, such as in leaves, stems and roots. Levels ofnon-polar lipids in vegetative plant parts can therefore be increased byreducing the activity of SDP1 polypeptides in the plant parts, forexample by either mutation of an endogenous gene encoding a SDP1polypeptide or introduction of an exogenous gene which encodes asilencing RNA molecule which reduces the expression of an endogenousSDP1 gene. Such a reduction is of particular benefit in tuber crops suchas sugarbeet and potato, and in “high sucrose” plants such as sugarcaneand and sugarbeet.

Genes encoding SDP1 homologues (including SDP1-like homologues) in aplant species of choice can be identified readily by homology to knownSDP1 gene sequences. Known SDP1 nucleotide or amino acid sequencesinclude Accession Nos.: in Brassica napus, GN078290 (SEQ ID NO:164),GN078281, GN078283; Capsella rubella, XP_006287072; Theobroma cacao,XP_007028574.1; Populus trichocarpa, XP_002308909 (SEQ ID NO:166);Prunus persica, XP_007203312; Prunus mume, XP_008240737; Malusdomestica, XP_008373034; Ricinus communis, XP_002530081; Medicagotruncatula, XP_003591425 (SEQ ID NO:167); Solanum lycopersicum,XP_004249208; Phaseolus vulgaris, XP_007162133; Glycine max,XP_003554141 (SEQ ID NO:168); Solanum tuberosum, XP_006351284; Glycinemax, XP_003521151; Cicer arietinum, XP_004493431; Cucumis sativus,XP_004142709; Cucumis melo, XP_008457586; Jatropha curcas, KDP26217;Vitis vinifera, CBI30074; Oryza sativa, Japonica Group BAB61223; Oryzasativa, Indica Group EAY75912; Oryza sativa, Japonica GroupNP_001044325; Sorghum bicolor, XP_002458531 (SEQ ID NO:169);Brachypodium distachyon, XP_003567139 (SEQ ID NO:165); Zea mays,AFW85009; Hordeum vulgare, BAK03290 (SEQ ID NO:172); Aegilops tauschii,EMT32802; Sorghum bicolor, XP_002463665; Zea mays. NP_001168677 (SEQ IDNO:170); Hordeum vulgare, BAK01155; Aegilops tauschii, EMT02623;Triticum urartu, EMS67257; Physcomitrella patens, XP_001758169 (SEQ IDNO:171). Preferred SDP1 sequences for use in genetic constructs forinhibiting expression of the endogenous genes are from cDNAscorresponding to the genes which are expressed most highly in the cells,vegetative plant parts or the seeds, whichever is to be modified.Nucleotide sequences which are highly conserved between cDNAscorresponding to all of the SDP1 genes in a plant species are preferredif it is desired to reduce the activity of all members of a gene familyin that species.

In an embodiment, a genetic modification of the invention down-regulatesendogenous production of SDP1, wherein SDP1 is encoded by one or more ofthe following:

i) nucleotides whose sequence is set forth as any one of SEQ ID NOs:163to 174,

ii) nucleotides whose sequence is at least 30% identical to any one ormore of the sequences set forth as SEQ ID NOs:163 to 174, and

iii) a sequence of nucleotides which hybridizes to one or both of i) orii) under stringent conditions.

As shown in the Examples, reduction of the expression and/or activity ofSDP1 TAG lipase in plant leaves greatly increased the TAG content, bothin terms of the amount of TAG that accumulated and the earlier timing ofaccumulation during plant development, in the context of co-expressionof the transcription factor WRI1 and a fatty acyl acyltransferase. Inparticular, the increase was observed in plants prior to flowering, andwas up to about 70% on a weight basis (% dry weight) at the onset ofsenescence. The increase was relative to the TAG levels observed incorresponding plant leaves transformed with exogenous polynucleotidesencoding the WRI1 and fatty acyl acyltransferase but lacking themodification that reduced SDP1 expression and/or activity.

Reducing the expression of other TAG catabolism genes in plant parts canalso increase TAG content, such as the ACX genes encoding acyl-CoAoxidases such as the Acx1 (At4g16760 and homologs in other plantspecies) or Acx2 (At5 g65110 and homologs in other plant species) genes.Another polypeptide involved in lipid catabolism is PXA1 which is aperoxisomal ATP-binding cassette transporter that is requires for fattyacid import for β-oxidation (Zolman et al. 2001).

Export of Fatty Acids from Plastids

As used herein, the term “polypeptide which increases the export offatty acids out of plastids of the cell” refers to any polypeptide whichaids in fatty acids being transferred from within plastids (in cellswhich have plastids such as a cell of a vegetative part, tuber, beet ora seed of a plant) to outside the plastid, which may be any other partof the cell such as for example the endoplasmic reticulum (ER). Examplesof such polypeptides include, but are not limited to, a C16 or C18 fattyacid thioesterase such as a FATA polypeptide or a FATB polypeptide, a C8to C14 fatty acid thioesterase (which is also a FATB polypeptide), afatty acid transporter such as an ABCA9 polypeptide or a long-chainacyl-CoA synthetase (LACS).

As used herein, the term “fatty acid thioesterase” or “FAT” refers to anenzyme which catalyses the hydrolysis of the thioester bond between anacyl moiety and acyl carrier protein (ACP) in acyl-ACP and the releaseof a free fatty acid. Such enzymes typically function in the plastids ofan organism which is synthesizing de novo fatty acids. As used herein,the term “C16 or C18 fatty acid thioesterase” refers to an enzyme whichcatalyses the hydrolysis of the thioester bond between a C16 and/or C18acyl moiety and ACP in acyl-ACP and the release of free C16 or C18 fattyacid in the plastid. The free fatty acid is then re-esterified to CoA inthe plastid envelope as it is transported out of the plastid. Thesubstrate specificity of the fatty acid thioesterase (FAT) enzyme in theplastid is involved in determining the spectrum of chain length anddegree of saturation of the fatty acids exported from the plastid. FATenzymes can be classified into two classes based on their substratespecificity and nucleotide sequences, FATA and FATB (EC 3.1.2.14) (Joneset al., 1995). FATA polypeptides prefer oleoyl-ACP as substrate, whileFATB polypeptides show higher activity towards saturated acyl-ACPs ofdifferent chain lengths such as acting on palmitoyl-ACP to produce freepalmitic acid. Examples of FATA polypeptides useful for the inventioninclude, but are not limited to, those from Arabidopsis thaliana(NP_189147), Arachis hypogaea (GU324446), Helianthus annuus (AAL79361),Carthamus tinctorius (AAA33020), Morus notabilis (XP_010104178.1),Brassica napus (CDX77369.1), Ricinus communis (XP_002532744.1) andCamelina sativa (AFQ60946.1). Examples of FATB polypeptides useful forthe invention include, but are not limited to, those from Zea mays(AIL28766), Brassica napus (ABHI 1710), Helianthus annuus (AAX 19387),Arabidopsis thaliana (AEE28300), Umbellularia californica (AAC49001),Arachis hypogaea (AFR54500), Ricinus communis (EEF47013) andBrachypodium sylvaticum (ABL85052.1).

A subclass of FATB polypeptides are fatty acid thioesterases which havehydrolysis activity on a C8-C14 saturated acyl moiety linked by athioester bond to ACP. Such enzymes are also referred to as medium chainfatty acid (MCFA) thioesterases or MC-FAT enzymes. Such enzymes may alsohave thioesterase activity on C16-ACP, indeed they may have greaterthioesterase activity on a C16 acyl-ACP substrate than on a MCFA-ACPsubstrate, nevertheless they are considered herein to be an MCFAthioesterase if they produce at least 0.5% MCFA in the total fatty acidcontent when expressed exogenously in a plant cell. Examples of MCFAthioesterases are given in Example 9 herein.

As used herein, the term “fatty acid transporter” relates to apolypeptide present in the plastid membrane which is involved inactively transferring fatty acids from a plastid to outside the plastid.Examples of ABCA9 (ABC transporter A family member 9) polypeptidesuseful for the invention include, but are not limited to, those fromArabidopsis thaliana (Q9FLT5), Capsella rubella (XP_006279962.1), Arabisalpine (KFK27923.1), Camelina sativa (XP_010457652.1), Brassica napus(CDY23040.1) and Brassica rapa (XP_009136512.1).

As used herein, the term “acyl-CoA synthetase” or “ACS” (EC 6.2.1.3)means a polypeptide which is a member of a ligase family that catalyzesthe formation of fatty acyl-CoA by a two-step process proceeding throughan adenylated intermediate, using a non-esterified fatty acid, CoA andATP as substrates to produce an acyl-CoA ester, AMP and pyrophosphate asproducts. As used herein, the term “long-chain acyl-CoA synthetase”(LACS) is an ACS that has activity on at least a C18 free fatty acidsubstrate although it may have broader activity on any of C14-C20 freefatty acids. The endogenous plastidial LACS enzymes are localised in theouter membrane of the plastid and function with fatty acid thioesterasefor the export of fatty acids from the plastid (Schnurr et al., 2002).In Arabidopsis, there are at least nine identified LACS genes (Shockeyet al., 2002). Preferred LACS polypeptides are of the LACS9 subclass,which in Arabidopsis is the major plastidial LACS. Examples of LACSpolypeptides useful for the invention include, but are not limited to,those from Arabidopsis thaliana (Q9CAP8), Camelina sativa(XP_010416710.1), Capsella rubella (XP_006301059.1), Brassica napus(CDX79212.1), Brassica rapa (XP_009104618.1), Gossypium raimondii(XP_012450538.1) and Vitis Vinifera (XP_002285853.1). Homologs of theabove mentioned polypeptides in other species can readily be identifiedby those skilled in the art.

Polypeptides Involved in Diacylglycerol (DAG) Production in Plastids

Levels of non-polar lipids in, for example, vegetative plant parts canalso be increased by reducing the activity of polypeptides involved indiacylglycerol (DAG) production in the plastid in the plant parts, forexample by either mutation of an endogenous gene encoding such apolypeptide or introduction of an exogenous gene which encodes asilencing RNA molecule which reduces the expression of a target geneinvolved in diacylglycerol (DAG) production in the plastid.

As used herein, the term “polypeptide involved in diacylglycerol (DAG)production in the plastid” refers to any polypeptide in the plastid (incells which have plastids such as a cell of a vegetative part, tuber,beet or a seed of a plant) that is directly involved in the synthesis ofdiacylglycerol. Examples of such polypeptides include, but are notlimited to, a plastidial GPAT, a plastidial LPAAT or a plastidial PAP.

GPATs are described elsewhere in the present document. Examples ofplastidial GPAT polypeptides which can be targeted for down-regulationin the invention include, but are not limited to, those from Arabidopsisthaliana (BAA00575), Capsella rubella (XP_006306544.1), Camelina sativa(010499766.1), Brassica napus (CDY43010.1), Brassica rapa(XP_009145198.1), Helianthus annuus (ADV16382.1) and Citrus unshiu(BAB79529.1). Homologs in other species can readily be identified bythose skilled in the art.

LPAATs are described elsewhere in the present document. As the skilledperson would appreciate, plastidial LPAATs to be targeted fordown-regulation for reducing DAG synthesis in the plastid are notendogenous LPAATs which function outside of the plastid such as those inthe ER, for example as described herein as being useful for producingTAG comprising medium chain length fatty acids. Examples of plastidialLPAAT polypeptides which can be targeted for down-regulation in theinvention include, but are not limited to, those from Brassica napus(ABQ42862), Brassica rapa (XP_009137939.1), Arabidopsis thaliana(NP_194787.2), Camelina sativa (XP_010432969.1), Glycine max(XP_006592638.1) and Solanum tuberosum (XP_006343651.1). Homologs inother species of the above mentioned polypeptides can readily beidentified by those skilled in the art.

As used herein, the term “phosphatidic acid phosphatase” (PAP) (EC3.1.3.4) refers to a protein which hydrolyses the phosphate group on3-sn-phosphatidate to produce 1,2-diacyl-sn-glycerol (DAG) andphosphate. Examples of plastidial PAP polypeptides which can be targetedfor down-regulation in the invention include, but are not limited to,those from Arabidopsis thaliana (Q6NLA5), Capsella rubella(XP_006288605.1), Camelina sativa (XP_010452170.1), Brassica napus(CDY10405.1), Brassica rapa (XP_009122733.1), Glycine max(XP_003542504.1) and Solanum tuberosum (XP_006361792.1). Homologs inother species of the above mentioned polypeptides can readily beidentified by those skilled in the art.

Import of Fatty Acids into Plastids

Levels of non-polar lipids in vegetative plant parts can also beincreased by reducing the activity of TGD polypeptides in the plantparts, for example by either mutation of an endogenous gene encoding aTGD polypeptide or introduction of an exogenous gene which encodes asilencing RNA molecule which reduces the expression of an endogenous TGDgene. As used herein, a “Trigalactosyldiacylglycerol (TGD) polypeptide”is one which is involved in the ER to chloroplast lipid trafficking (Xuet al., 2010) and involved in forming a protein complex which haspermease function for lipids. Four such polypeptides are known to formor be associated with a TGD permease, namely TGD-1 (Accession No.At1g19800 and homologs in other species), TGD-2 (Accession No At2 g20320and homologs in other species), TGD-3 (Accession No. NM-105215 andhomologs in other species) and TGD-4 (At3 g06960 and homologs in otherspecies) (US 20120237949). TGD-1, -2 and -3 polypeptides are thought tobe components of an ATP-Binding Cassette (ABC) transporter associatedwith the inner envelope membrane of the chloroplast. TGD-2 and TGD-4polypeptides bind to phosphatidic acid whereas TGD-3 polypetidefunctions as an ATPase in the chloroplast stroma. As used herein, an“endogenous TGD gene” is a gene which encodes a TGD polypeptide in aplant. Mutations in TGD-1 gene in A. thaliana caused accumulation oftriacylglycerols, oligogalactolipids and phosphatidic acid (PA) (Xu etal., 2005). Mutations in TGD genes or SDP1 genes, or indeed in anydesired gene in a plant, can be introduced in a site-specific manner byartificial zinc finger nuclease (ZFN), TAL effector (TALEN) or CRISPRtechnologies (using a Cas9 type nuclease) as known in the art. Preferredexogenous genes encoding silencing RNAs are those encoding adouble-stranded RNA molecule such as a hairpin RNA or an artificialmicroRNA precursor.

Fatty Acid Modifying Enzymes

As used herein, the term “FAD2” refers to a membrane bound delta-12fatty acid desturase that desaturates oleic acid (C18:1^(Δ9)) to producelinoleic acid (C18:2^(Δ9,12)).

As used herein, the term “epoxygenase” or “fatty acid epoxygenase”refers to an enzyme that introduces an epoxy group into a fatty acidresulting in the production of an epoxy fatty acid. In preferredembodiment, the epoxy group is introduced at the 12th carbon on a fattyacid chain, in which case the epoxygenase is a Δ12-epoxygenase,especially of a C16 or C18 fatty acid chain. The epoxygenase may be aΔ9-epoxygenase, a Δ15 epoxygenase, or act at a different position in theacyl chain as known in the art. The epoxygenase may be of the P450class. Preferred epoxygenases are of the mono-oxygenase class asdescribed in WO98/46762. Numerous epoxygenases or presumed epoxygenaseshave been cloned and are known in the art. Further examples ofexpoxygenases include proteins comprising an amino acid sequenceprovided in SEQ ID NO:21 of WO 2009/129582, polypeptides encoded bygenes from Crepis paleastina (CAA76156, Lee et al., 1998), Stokesialaevis (AAR23815) (monooxygenase type), Euphorbia lagascae (AAL62063)(P450 type), human CYP2J2 (arachidonic acid epoxygenase, U37143); humanCYPIA1 (arachidonic acid epoxygenase, K03191), as well as variantsand/or mutants thereof.

As used herein, the term, “hydroxylase” or “fatty acid hydroxylase”refers to an enzyme that introduces a hydroxyl group into a fatty acidresulting in the production of a hydroxylated fatty acid. In a preferredembodiment, the hydroxyl group is introduced at the 2nd, 12th and/or17th carbon on a C18 fatty acid chain. Preferably, the hydroxyl group isintroduced at the 12^(th) carbon, in which case the hydroxylase is aΔ12-hydroxylase. In another preferred embodiment, the hydroxyl group isintroduced at the 15th carbon on a C16 fatty acid chain. Hydroxylasesmay also have enzyme activity as a fatty acid desaturase. Examples ofgenes encoding Δ12-hydroxylases include those from Ricinus communis(AAC9010, van de Loo 1995); Physaria lindheimeri, (ABQ01458, Dauk etal., 2007); Lesquerella fendleri, (AAC32755, Broun et al., 1998); Daucuscarota, (AAK30206); fatty acid hydroxylases which hydroxylate theterminus of fatty acids, for example: A. thaliana CYP86A1 (P48422, fattyacid (ω-hydroxylase); Vicia sativa CYP94A1 (P98188, fatty acidω-hydroxylase); mouse CYP2E1 (X62595, lauric acid ω-1 hydroxylase); ratCYP4A1 (M57718, fatty acid ω-hydroxylase), as well as as variants and/ormutants thereof.

As used herein, the term “conjugase” or “fatty acid conjugase” refers toan enzyme capable of forming a conjugated bond in the acyl chain of afatty acid. Examples of conjugases include those encoded by genes fromCalendula officinalis (AF343064, Qiu et al., 2001); Vernicia fordii(AAN87574, Dyer et al., 2002); Punica granatum (AY178446, Iwabuchi etal., 2003) and Trichosanthes kirilowii (AY178444, Iwabuchi et al.,2003); as well as as variants and/or mutants thereof.

As used herein, the term “acetylenase” or “fatty acid acetylenase”refers to an enzyme that introduces a triple bond into a fatty acidresulting in the production of an acetylenic fatty acid. In a preferredembodiment, the triple bond is introduced at the 2nd, 6th, 12th and/or17th carbon on a C18 fatty acid chain. Examples acetylenases includethose from Helianthus annuus (AA038032, ABC59684), as well as asvariants and/or mutants thereof.

Examples of such fatty acid modifying genes include proteins accordingto the following Accession Numbers which are grouped by putativefunction, and homologues from other species: Δ12-acetylenases ABC00769,CAA76158, AAO38036, AAO38032; Δ12 conjugases AAG42259, AAG42260,AAN87574; Δ12-desaturases P46313, ABS18716, AAS57577, AAL61825,AAF04093, AAF04094; Δ12 epoxygenases XP_001840127, CAA76156, AAR23815;Δ12-hydroxylases ACF37070, AAC32755, ABQ01458, AAC49010; and Δ12 P450enzymes such as AF406732.

Silencing Suppressors

In an embodiment, a recombinant/transgenic cell of the invention maycomprise a silencing suppressor.

As used herein, a “silencing suppressor” enhances transgene expressionin a cell of the invention. For example, the presence of the silencingsuppressor results in higher levels of a polypeptide(s) produced anexogenous polynucleotide(s) in a cell of the invention when compared toa corresponding cell lacking the silencing suppressor. In an embodiment,the silencing suppressor preferentially binds a dsRNA molecule which is21 base pairs in length relative to a dsRNA molecule of a differentlength. This is a feature of at least the p19 type of silencingsuppressor, namely for p19 and its functional orthologs. In anotherembodiment, the silencing suppressor preferentially binds to adouble-stranded RNA molecule which has overhanging 5′ ends relative to acorresponding double-stranded RNA molecule having blunt ends. This is afeature of the V2 type of silencing suppressor, namely for V2 and itsfunctional orthologs. In an embodiment, the dsRNA molecule, or aprocessed RNA product thereof, comprises at least 19 consecutivenucleotides, preferably whose length is 19-24 nucleotides with 19-24consecutive basepairs in the case of a double-stranded hairpin RNAmolecule or processed RNA product, more preferably consisting of 20, 21,22, 23 or 24 nucleotides in length, and preferably comprising amethylated nucleotide, which is at least 95% identical to the complementof the region of the target RNA, and wherein the region of the targetRNA is i) within a 5′ untranslated region of the target RNA, ii) withina 5′ half of the target RNA, iii) within a protein-encoding open-readingframe of the target RNA, iv) within a 3′ half of the target RNA, or v)within a 3′ untranslated region of the target RNA.

Further details regarding silencing suppressors are well known in theart and described in WO 2013/096992 and WO 2013/096993.

Polynucleotides

The terms “polynucleotide”, and “nucleic acid” are used interchangeably.They refer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof. Apolynucleotide of the invention may be of genomic, cDNA, semisynthetic,or synthetic origin, double-stranded or single-stranded and by virtue ofits origin or manipulation: (1) is not associated with all or a portionof a polynucleotide with which it is associated in nature, (2) is linkedto a polynucleotide other than that to which it is linked in nature, or(3) does not occur in nature. The following are non-limiting examples ofpolynucleotides: coding or non-coding regions of a gene or genefragment, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA),ribosomal RNA (rRNA), ribozymes, cDNA, recombinant polynucleotides,plasmids, vectors, isolated DNA of any sequence, isolated RNA of anysequence, chimeric DNA of any sequence, nucleic acid probes, andprimers. For in vitro use, a polynucleotide may comprise modifiednucleotides such as by conjugation with a labeling component.

As used herein, an “isolated polynucleotide” refers to a polynucleotidewhich has been separated from the polynucleotide sequences with which itis associated or linked in its native state, or a non-naturallyoccurring polynucleotide.

As used herein, the term “gene” is to be taken in its broadest contextand includes the deoxyribonucleotide sequences comprising thetranscribed region and, if translated, the protein coding region, of astructural gene and including sequences located adjacent to the codingregion on both the 5′ and 3′ ends for a distance of at least about 2 kbon either end and which are involved in expression of the gene. In thisregard, the gene includes control signals such as promoters, enhancers,termination and/or polyadenylation signals that are naturally associatedwith a given gene, or heterologous control signals, in which case, thegene is referred to as a “chimeric gene”. The sequences which arelocated 5′ of the protein coding region and which are present on themRNA are referred to as 5′ non-translated sequences. The sequences whichare located 3′ or downstream of the protein coding region and which arepresent on the mRNA are referred to as 3′ non-translated sequences. Theterm “gene” encompasses both cDNA and genomic forms of a gene. A genomicform or clone of a gene contains the coding region which may beinterrupted with non-coding sequences termed “introns”, “interveningregions”. or “intervening sequences.” Introns are segments of a genewhich are transcribed into nuclear RNA (nRNA). Introns may containregulatory elements such as enhancers. Introns are removed or “splicedout” from the nuclear or primary transcript; introns are thereforeabsent in the mRNA transcript. A gene which contains at least one intronmay be subject to variable splicing, resulting in alternative mRNAs froma single transcribed gene and therefore polypeptide variants. A gene inits native state, or a chimeric gene may lack introns. The mRNAfunctions during translation to specify the sequence or order of aminoacids in a nascent polypeptide. The term “gene” includes a synthetic orfusion molecule encoding all or part of the proteins of the inventiondescribed herein and a complementary nucleotide sequence to any one ofthe above.

As used herein, “chimeric DNA” refers to any DNA molecule that is notnaturally found in nature; also referred to herein as a “DNA construct”or “genetic construct”. Typically, a chimeric DNA comprises regulatoryand transcribed or protein coding sequences that are not naturally foundtogether in nature. Accordingly, chimeric DNA may comprise regulatorysequences and coding sequences that are derived from different sources,or regulatory sequences and coding sequences derived from the samesource, but arranged in a manner different than that found in nature.The open reading frame may or may not be linked to its natural upstreamand downstream regulatory elements. The open reading frame may beincorporated into, for example, the plant genome, in a non-naturallocation, or in a replicon or vector where it is not naturally foundsuch as a bacterial plasmid or a viral vector. The term “chimeric DNA”is not limited to DNA molecules which are replicable in a host, butincludes DNA capable of being ligated into a replicon by, for example,specific adaptor sequences.

A “transgene” is a gene that has been introduced into the genome by atransformation procedure. The term includes a gene in a progeny cell,plant, seed, non-human organism or part thereof which was introducinginto the genome of a progenitor cell thereof. Such progeny cells etc maybe at least a 3^(rd) or 4^(th) generation progeny from the progenitorcell which was the primary transformed cell, or of the progenitortransgenic plant (referred to herein as a T0 plant). Progeny may beproduced by sexual reproduction or vegetatively such as, for example,from tubers in potatoes or ratoons in sugarcane. The term “geneticallymodified”, “genetic modification” and variations thereof, is a broaderterm that includes introducing a gene into a cell by transformation ortransduction, mutating a gene in a cell and genetically altering ormodulating the regulation of a gene in a cell, or the progeny of anycell modified as described above.

A “genomic region” as used herein refers to a position within the genomewhere a transgene, or group of transgenes (also referred to herein as acluster), have been inserted into a cell, or predecessor thereof. Suchregions only comprise nucleotides that have been incorporated by theintervention of man such as by methods described herein.

A “recombinant polynucleotide” of the invention refers to a nucleic acidmolecule which has been constructed or modified by artificialrecombinant methods. The recombinant polynucleotide may be present in acell in an altered amount or expressed at an altered rate (e.g., in thecase of mRNA) compared to its native state. In one embodiment, thepolynucleotide is introduced into a cell that does not naturallycomprise the polynucleotide. Typically an exogenous DNA is used as atemplate for transcription of mRNA which is then translated into acontinuous sequence of amino acid residues coding for a polypeptide ofthe invention within the transformed cell. In another embodiment, thepolynucleotide is endogenous to the cell and its expression is alteredby recombinant means, for example, an exogenous control sequence isintroduced upstream of an endogenous gene of interest to enable thetransformed cell to express the polypeptide encoded by the gene, or adeletion is created in a gene of interest by ZFN, Talen or CRISPRmethods.

A recombinant polynucleotide of the invention includes polynucleotideswhich have not been separated from other components of the cell-based orcell-free expression system, in which it is present, and polynucleotidesproduced in said cell-based or cell-free systems which are subsequentlypurified away from at least some other components. The polynucleotidecan be a contiguous stretch of nucleotides or comprise two or morecontiguous stretches of nucleotides from different sources (naturallyoccurring and/or synthetic) joined to form a single polynucleotide.Typically, such chimeric polynucleotides comprise at least an openreading frame encoding a polypeptide of the invention operably linked toa promoter suitable of driving transcription of the open reading framein a cell of interest.

With regard to the defined polynucleotides, it will be appreciated that% identity figures higher than those provided above will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polynucleotide comprises apolynucleotide sequence which is at least 60%, more preferably at least65%, more preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, more preferably at least 93%, more preferably at least 94%, morepreferably at least 95%, more preferably at least 96%, more preferablyat least 97%, more preferably at least 98%, more preferably at least99%, more preferably at least 99.1%, more preferably at least 99.2%,more preferably at least 99.3%, more preferably at least 99.4%, morepreferably at least 99.5%, more preferably at least 99.6%, morepreferably at least 99.7%, more preferably at least 99.8%, and even morepreferably at least 99.9% identical to the relevant nominated SEQ ID NO.

A polynucleotide of, or useful for, the present invention mayselectively hybridise, under stringent conditions, to a polynucleotidedefined herein. As used herein, stringent conditions are those that: (1)employ during hybridisation a denaturing agent such as formamide, forexample, 50% (v/v) formamide with 0.1% (w/v) bovine serum albumin, 0.1%Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C.; or (2) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 g/ml), 0.1% SDS and 10% dextran sulfateat 42° C. in 0.2×SSC and 0.1% SDS, and/or (3) employ low ionic strengthand high temperature for washing, for example, 0.015 M NaCl/0.0015 Msodium citrate/0.1% SDS at 50° C.

Polynucleotides of the invention may possess, when compared to naturallyoccurring molecules, one or more mutations which are deletions,insertions, or substitutions of nucleotide residues. Polynucleotideswhich have mutations relative to a reference sequence can be eithernaturally occurring (that is to say, isolated from a natural source) orsynthetic (for example, by performing site-directed mutagenesis or DNAshuffling on the nucleic acid as described above).

Polynucleotides for Reducing Expression of Genes

RNA Interference

RNA interference (RNAi) is particularly useful for specifically reducingthe expression of a gene, which results in reduced production of aparticular protein if the gene encodes a protein. Although not wishingto be limited by theory, Waterhouse et al. (1998) have provided a modelfor the mechanism by which dsRNA (duplex RNA) can be used to reduceprotein production. This technology relies on the presence of dsRNAmolecules that contain a sequence that is essentially identical to themRNA of the gene of interest or part thereof. Conveniently, the dsRNAcan be produced from a single promoter in a recombinant vector or hostcell, where the sense and anti-sense sequences are flanked by anunrelated sequence which enables the sense and anti-sense sequences tohybridize to form the dsRNA molecule with the unrelated sequence forminga loop structure. The design and production of suitable dsRNA moleculesis well within the capacity of a person skilled in the art, particularlyconsidering Waterhouse et al. (1998), Smith et al. (2000), WO 99/32619,WO 99/53050, WO 99/49029, and WO 01/34815.

In one example, a DNA is introduced that directs the synthesis of an atleast partly double stranded RNA product(s) with homology to the targetgene to be inactivated such as, for example, a SDP1, TGD, plastidialGPAT, plastidial LPAAT, plastidial PAP, AGPase gene. The DNA thereforecomprises both sense and antisense sequences that, when transcribed intoRNA, can hybridize to form the double stranded RNA region. In oneembodiment of the invention, the sense and antisense sequences areseparated by a spacer region that comprises an intron which, whentranscribed into RNA, is spliced out. This arrangement has been shown toresult in a higher efficiency of gene silencing (Smith et al., 2000).The double stranded region may comprise one or two RNA molecules,transcribed from either one DNA region or two. The presence of thedouble stranded molecule is thought to trigger a response from anendogenous system that destroys both the double stranded RNA and alsothe homologous RNA transcript from the target gene, efficiently reducingor eliminating the activity of the target gene.

The length of the sense and antisense sequences that hybridize shouldeach be at least 19 contiguous nucleotides, preferably at least 50contiguous nucleotides, more preferably at least 100 or at least 200contiguous nucleotides. Generally, a sequence of 100-1000 nucleotidescorresponding to a region of the target gene mRNA is used. Thefull-length sequence corresponding to the entire gene transcript may beused. The degree of identity of the sense sequence to the targetedtranscript (and therefore also the identity of the antisense sequence tothe complement of the target transcript) should be at least 85%, atleast 90%, or 95-100%. The RNA molecule may of course comprise unrelatedsequences which may function to stabilize the molecule. The RNA moleculemay be expressed under the control of a RNA polymerase II or RNApolymerase III promoter. Examples of the latter include tRNA or snRNApromoters.

Preferred small interfering RNA (“siRNA”) molecules comprise anucleotide sequence that is identical to about 19-25 contiguousnucleotides of the target mRNA. Preferably, the siRNA sequence commenceswith the dinucleotide AA, comprises a GC-content of about 30-70%(preferably, 30-60%, more preferably 40-60% and more preferably about45%-55%), and does not have a high percentage identity to any nucleotidesequence other than the target in the genome of the organism in which itis to be introduced, for example, as determined by standard BLASTsearch.

microRNA

MicroRNAs (abbreviated miRNAs) are generally 19-25 nucleotides (commonlyabout 20-24 nucleotides in plants) non-coding RNA molecules that arederived from larger precursors that form imperfect stem-loop structures.miRNAs bind to complementary sequences on target messenger RNAtranscripts (mRNAs), usually resulting in translational repression ortarget degradation and gene silencing. Artificial miRNAs (amiRNAs) canbe designed based on natural miRNAs for reducing the expression of anygene of interest, as well known in the art.

In plant cells, miRNA precursor molecules are believed to be largelyprocessed in the nucleus. The pri-miRNA (containing one or more localdouble-stranded or “hairpin” regions as well as the usual 5′ “cap” andpolyadenylated tail of an mRNA) is processed to a shorter miRNAprecursor molecule that also includes a stem-loop or fold-back structureand is termed the “pre-miRNA”. In plants, the pre-miRNAs are cleaved bydistinct DICER-like (DCL) enzymes, yielding miRNA:miRNA*duplexes. Priorto transport out of the nucleus, these duplexes are methylated.

In the cytoplasm, the miRNA strand from the miRNA:miRNA duplex isselectively incorporated into an active RNA-induced silencing complex(RISC) for target recognition. The RISC-complexes contain a particularsubset of Argonaute proteins that exert sequence-specific generepression (see, for example, Millar and Waterhouse, 2005; Pasquinelliet al., 2005; Almeida and Allshire, 2005).

Cosuppression

Genes can suppress the expression of related endogenous genes and/ortransgenes already present in the genome, a phenomenon termedhomology-dependent gene silencing. Most of the instances ofhomologydependent gene silencing fall into two classes—those thatfunction at the level of transcription of the transgene, and those thatoperate post-transcriptionally.

Post-transcriptional homology-dependent gene silencing (i.e.,cosuppression) describes the loss of expression of a transgene andrelated endogenous or viral genes in transgenic plants. Cosuppressionoften, but not always, occurs when transgene transcripts are abundant,and it is generally thought to be triggered at the level of mRNAprocessing, localization, and/or degradation. Several models exist toexplain how cosuppression works (see in Taylor, 1997).

Cosuppression involves introducing an extra copy of a gene or a fragmentthereof into a plant in the sense orientation with respect to a promoterfor its expression. The size of the sense fragment, its correspondenceto target gene regions, and its degree of sequence identity to thetarget gene can be determined by those skilled in the art. In someinstances, the additional copy of the gene sequence interferes with theexpression of the target plant gene. Reference is made to WO 97/20936and EP 0465572 for methods of implementing co-suppression approaches.

Antisense Polynucleotides

The term “antisense polynucletoide” shall be taken to mean a DNA or RNAmolecule that is complementary to at least a portion of a specific mRNAmolecule encoding an endogenous polypeptide and capable of interferingwith a post-transcriptional event such as mRNA translation. The use ofantisense methods is well known in the art (see for example, G. Hartmannand S. Endres, Manual of Antisense Methodology, Kluwer (1999)). The useof antisense techniques in plants has been reviewed by Bourque (1995)and Senior (1998). Bourque (1995) lists a large number of examples ofhow antisense sequences have been utilized in plant systems as a methodof gene inactivation. Bourque also states that attaining 100% inhibitionof any enzyme activity may not be necessary as partial inhibition willmore than likely result in measurable change in the system. Senior(1998) states that antisense methods are now a very well establishedtechnique for manipulating gene expression.

In one embodiment, the antisense polynucleotide hybridises underphysiological conditions, that is, the antisense polynucleotide (whichis fully or partially single stranded) is at least capable of forming adouble stranded polynucleotide with mRNA encoding an endogenouspolypeptide, for example, a SDP1, TGD, plastidial GPAT, plastidialLPAAT, plastidial PAP or AGPase mRNA under normal conditions in a cell.

Antisense molecules may include sequences that correspond to thestructural genes or for sequences that effect control over the geneexpression or splicing event. For example, the antisense sequence maycorrespond to the targeted coding region of endogenous gene, or the5′-untranslated region (UTR) or the 3′-UTR or combination of these. Itmay be complementary in part to intron sequences, which may be splicedout during or after transcription, preferably only to exon sequences ofthe target gene. In view of the generally greater divergence of theUTRs, targeting these regions provides greater specificity of geneinhibition.

The length of the antisense sequence should be at least 19 contiguousnucleotides, preferably at least 50 nucleotides, and more preferably atleast 100, 200, 500 or 1000 nucleotides. The full-length sequencecomplementary to the entire gene transcript may be used. The length ismost preferably 100-2000 nucleotides. The degree of identity of theantisense sequence to the targeted transcript should be at least 90% andmore preferably 95-100%. The antisense RNA molecule may of coursecomprise unrelated sequences which may function to stabilize themolecule.

Recombinant Vectors

One embodiment of the present invention includes a recombinant vector,which comprises at least one polynucleotide defined herein and iscapable of delivering the polynucleotide into a host cell. Recombinantvectors include expression vectors. Recombinant vectors containheterologous polynucleotide sequences, that is, polynucleotide sequencesthat are not naturally found adjacent to a polynucleotide definedherein, that preferably, are derived from a different species. Thevector can be either RNA or DNA, and typically is a viral vector,derived from a virus, or a plasmid. Plasmid vectors typically includeadditional nucleic acid sequences that provide for easy selection,amplification, and transformation of the expression cassette inprokaryotic cells, e.g., pUC-derived vectors, pGEM-derived vectors orbinary vectors containing one or more T-DNA regions. Additional nucleicacid sequences include origins of replication to provide for autonomousreplication of the vector, selectable marker genes, preferably encodingantibiotic or herbicide resistance, unique multiple cloning sitesproviding for multiple sites to insert nucleic acid sequences or genesencoded in the nucleic acid construct, and sequences that enhancetransformation of prokaryotic and eukaryotic (especially plant) cells.

“Operably linked” as used herein, refers to a functional relationshipbetween two or more nucleic acid (e.g., DNA) segments. Typically, itrefers to the functional relationship of a transcriptional regulatoryelement (promoter) to a transcribed sequence. For example, a promoter isoperably linked to a coding sequence of a polynucleotide defined herein,if it stimulates or modulates the transcription of the coding sequencein an appropriate cell. Generally, promoter transcriptional regulatoryelements that are operably linked to a transcribed sequence arephysically contiguous to the transcribed sequence, i.e., they arecis-acting. However, some transcriptional regulatory elements such asenhancers need not be physically contiguous or located in closeproximity to the coding sequences whose transcription they enhance.

When there are multiple promoters present, each promoter mayindependently be the same or different.

Recombinant vectors may also contain one or more signal peptidesequences to enable an expressed polypeptide defined herein to beretained in the endoplasmic reticulum (ER) in the cell, or transfer intoa plastid, and/or contain fusion sequences which lead to the expressionof nucleic acid molecules as fusion proteins. Examples of suitablesignal segments include any signal segment capable of directing thesecretion or localisation of a polypeptide defined herein.

To facilitate identification of transformants, the recombinant vectordesirably comprises a selectable or screenable marker gene. By “markergene” is meant a gene that imparts a distinct phenotype to cellsexpressing the marker gene and thus, allows such transformed cells to bedistinguished from cells that do not have the marker. A selectablemarker gene confers a trait for which one can “select” based onresistance to a selective agent (e.g., a herbicide, antibiotic). Ascreenable marker gene (or reporter gene) confers a trait that one canidentify through observation or testing, that is, by “screening” (e.g.,β-glucuronidase, luciferase, GFP or other enzyme activity not present inuntransformed cells). Exemplary selectable markers for selection ofplant transformants include, but are not limited to, a hyg gene whichencodes hygromycin B resistance; a neomycin phosphotransferase (nptII)gene conferring resistance to kanamycin, paromomycin; aglutathione-S-transferase gene from rat liver conferring resistance toglutathione derived herbicides as for example, described in EP 256223; aglutamine synthetase gene conferring, upon overexpression, resistance toglutamine synthetase inhibitors such as phosphinothricin as for example,described in WO 87/05327; an acetyltransferase gene from Streptomycesviridochromogenes conferring resistance to the selective agentphosphinothricin as for example, described in EP 275957; a gene encodinga 5-enolshikimate-3-phosphate synthase (EPSPS) conferring tolerance toN-phosphonomethylglycine as for example, described by Hinchee et al.(1988); a bar gene conferring resistance against bialaphos as forexample, described in WO91/02071; a nitrilase gene such as bxn fromKlebsiella ozaenae which confers resistance to bromoxynil (Stalker etal., 1988); a dihydrofolate reductase (DHFR) gene conferring resistanceto methotrexate (Thillet et al., 1988); a mutant acetolactate synthasegene (ALS) which confers resistance to imidazolinone, sulfonylurea, orother ALS-inhibiting chemicals (EP 154,204); a mutated anthranilatesynthase gene that confers resistance to 5-methyl tryptophan; or adalapon dehalogenase gene that confers resistance to the herbicide.

Preferably, the recombinant vector is stably incorporated into thegenome of the cell such as the plant cell. Accordingly, the recombinantvector may comprise appropriate elements which allow the vector to beincorporated into the genome, or into a chromosome of the cell.

Expression Vector

As used herein, an “expression vector” is a DNA vector that is capableof transforming a host cell and of effecting expression of one or morespecified polynucleotides. Expression vectors of the present inventioncontain regulatory sequences such as transcription control sequences,translation control sequences, origins of replication, and otherregulatory sequences that are compatible with the host cell and thatcontrol the expression of polynucleotides of the present invention. Inparticular, expression vectors of the present invention includetranscription control sequences. Transcription control sequences aresequences which control the initiation, elongation, and termination oftranscription. Particularly important transcription control sequencesare those which control transcription initiation such as promoter,enhancer, operator and repressor sequences. The choice of the regulatorysequences used depends on the target organism such as a plant and/ortarget organ or tissue of interest. Such regulatory sequences may beobtained from any eukaryotic organism such as plants or plant viruses,or may be chemically synthesized. A number of vectors suitable forstable transfection of plant cells or for the establishment oftransgenic plants have been described in for example, Pouwels et al.,Cloning Vectors: A Laboratory Manual, 1985, supp. 1987, Weissbach andWeissbach, Methods for Plant Molecular Biology, Academic Press, 1989,and Gelvin et al., Plant Molecular Biology Manual, Kluwer AcademicPublishers, 1990. Typically, plant expression vectors include forexample, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, a transcription termination site, and/or apolyadenylation signal.

A number of constitutive promoters that are active in plant cells havebeen described. Suitable promoters for constitutive expression in plantsinclude, but are not limited to, the cauliflower mosaic virus (CaMV) 35Spromoter, the Figwort mosaic virus (FMV) 35S, the light-induciblepromoter from the small subunit (SSU) of the ribulose-1,5-bis-phosphatecarboxylase, the rice cytosolic triosephosphate isomerase promoter, theadenine phosphoribosyltransferase promoter of Arabidopsis, the riceactin 1 gene promoter, the mannopine synthase and octopine synthasepromoters, the Adh promoter, the sucrose synthase promoter, the R genecomplex promoter, and the chlorophyll α/β binding protein gene promoter.These promoters have been used to create DNA vectors that have beenexpressed in plants, see for example, WO 84/02913. All of thesepromoters have been used to create various types of plant-expressiblerecombinant DNA vectors.

For the purpose of expression in source tissues of the plant such as theleaf, seed, root or stem, it is preferred that the promoters utilized inthe present invention have relatively high expression in these specifictissues. For this purpose, one may choose from a number of promoters forgenes with tissue- or cell-specific, or -enhanced expression. Examplesof such promoters reported in the literature include, the chloroplastglutamine synthetase GS2 promoter from pea, the chloroplastfructose-1,6-biphosphatase promoter from wheat, the nuclearphotosynthetic ST-LS 1 promoter from potato, the serine/threonine kinasepromoter and the glucoamylase (CHS) prorhoter from Arabidopsis thaliana.Also reported to be active in photosynthetically active tissues are theribulose-1,5-bisphosphate carboxylase promoter from eastern larch (Larixlaricina), the promoter for the Cab gene, Cab6, from pine, the promoterfor the Cab-1 gene from wheat, the promoter for the Cab-1 gene fromspinach, the promoter for the Cab 1R gene from rice, the pyruvate,orthophosphate dikinase (PPDK) promoter from Zea mays, the promoter forthe tobacco Lhcb1*2 gene, the Arabidopsis thaliana Suc2 sucrose-H³⁰symporter promoter, and the promoter for the thylakoid membrane proteingenes from spinach (PsaD, PsaF, PsaE, PC, FNR, AtpC, AtpD, Cab, RbcS).Other promoters for the chlorophyll α/β-binding proteins may also beutilized in the present invention such as the promoters for LhcB geneand PsbP gene from white mustard (Sinapis alba).

A variety of plant gene promoters that are regulated in response toenvironmental, hormonal, chemical, and/or developmental signals, alsocan be used for expression of RNA-binding protein genes in plant cells,including promoters regulated by (1) heat, (2) light (e.g., pea RbcS-3Apromoter, maize RbcS promoter), (3) hormones such as abscisic acid, (4)wounding (e.g., WunI), or (5) chemicals such as methyl jasmonate,salicylic acid, steroid hormones, alcohol, Safeners (WO 97/06269), or itmay also be advantageous to employ (6) organ-specific promoters.

As used herein, the term “plant storage organ specific promoter” refersto a promoter that preferentially, when compared to other plant tissues,directs gene transcription in a storage organ of a plant. For thepurpose of expression in sink tissues of the plant such as the tuber ofthe potato plant, the fruit of tomato, or the seed of soybean, canola,cotton, Zea mays, wheat, rice, and barley, it is preferred that thepromoters utilized in the present invention have relatively highexpression in these specific tissues. The promoter for β-conglycinin orother seed-specific promoters such as the napin, zein, linin andphaseolin promoters, can be used. Root specific promoters may also beused. An example of such a promoter is the promoter for the acidchitinase gene. Expression in root tissue could also be accomplished byutilizing the root specific subdomains of the CaMV 35S promoter thathave been identified.

In a particularly preferred embodiment, the promoter directs expressionin tissues and organs in which lipid biosynthesis take place. Suchpromoters may act in seed development at a suitable time for modifyinglipid composition in seeds. Preferred promoters for seed-specificexpression include: 1) promoters from genes encoding enzymes involved inlipid biosynthesis and accumulation in seeds such as desaturases andelongases, 2) promoters from genes encoding seed storage proteins, and3) promoters from genes encoding enzymes involved in carbohydratebiosynthesis and accumulation in seeds. Seed specific promoters whichare suitable are, the oilseed rape napin gene promoter (U.S. Pat. No.5,608,152), the Vicia faba USP promoter (Baumlein et al., 1991), theArabidopsis oleosin promoter (WO 98/45461), the Phaseolus vulgarisphaseolin promoter (U.S. Pat. No. 5,504,200), the Brassica Bce4 promoter(WO 91/13980), or the legumin B4 promoter (Baumlein et al., 1992), andpromoters which lead to the seed-specific expression in monocots such asmaize, barley, wheat, rye, rice and the like. Notable promoters whichare suitable are the barley lpt2 or lpt1 gene promoter (WO 95/15389 andWO 95/23230), or the promoters described in WO 99/16890 (promoters fromthe barley hordein gene, the rice glutelin gene, the rice oryzin gene,the rice prolamin gene, the wheat gliadin gene, the wheat glutelin gene,the maize zein gene, the oat glutelin gene, the sorghum kasirin gene,the rye secalin gene). Other promoters include those described by Brounet al. (1998), Potenza et al. (2004), US 20070192902 and US 20030159173.In an embodiment, the seed specific promoter is preferentially expressedin defined parts of the seed such as the cotyledon(s) or the endosperm.Examples of cotyledon specific promoters include, but are not limitedto, the FP1 promoter (Ellerstrom et al., 1996), the pea legumin promoter(Perrin et al., 2000), and the bean phytohemagglutnin promoter (Perrinet al., 2000). Examples of endosperm specific promoters include, but arenot limited to, the maize zein-1 promoter (Chikwamba et al., 2003), therice glutelin-1 promoter (Yang et al., 2003), the barley D-hordeinpromoter (Horvath et al., 2000) and wheat HMW glutenin promoters(Alvarez et al., 2000). In a further embodiment, the seed specificpromoter is not expressed, or is only expressed at a low level, in theembryo and/or after the seed germinates.

In another embodiment, the plant storage organ specific promoter is afruit specific promoter. Examples include, but are not limited to, thetomato polygalacturonase, E8 and Pds promoters, as well as the apple ACCoxidase promoter (for review, see Potenza et al., 2004). In a preferredembodiment, the promoter preferentially directs expression in the edibleparts of the fruit, for example the pith of the fruit, relative to theskin of the fruit or the seeds within the fruit.

In an embodiment, the inducible promoter is the Aspergillus nidulans alcsystem. Examples of inducible expression systems which can be usedinstead of the Aspergillus nidulans alc system are described in a reviewby Padidam (2003) and Corrado and Karali (2009). In another embodiment,the inducible promoter is a safener inducible promoter such as, forexample, the maize ln2-1 or ln2-2 promoter (Hershey and Stoner, 1991),the safener inducible promoter is the maize GST-27 promoter (Jepson etal., 1994), or the soybean GH2/4 promoter (Ulmasov et al., 1995).

In another embodiment, the inducible promoter is a senescence induciblepromoter such as, for example, senescence-inducible promoter SAG(senescence associated gene) 12 and SAG13 from Arabidopsis (Gan, 1995;Gan and Amasino, 1995) and LSC54 from Brassica napus(Buchanan-Wollaston, 1994). Such promoters show increased expression atabout the onset of senescence of plant tissues, in particular theleaves.

For expression in vegetative tissue leaf-specific promoters, such as theribulose biphosphate carboxylase (RBCS) promoters, can be used. Forexample, the tomato RBCS1, RBCS2 and RBCS3A genes are expressed inleaves and light grown seedlings (Meier et al., 1997). A ribulosebisphosphate carboxylase promoters expressed almost exclusively inmesophyll cells in leaf blades and leaf sheaths at high levels,described by Matsuoka et al. (1994), can be used. Another leaf-specificpromoter is the light harvesting chlorophyll a/b binding protein genepromoter (see, Shiina et al., 1997). The Arabidopsis thalianamyb-related gene promoter (Atmyb5) described by Li et al. (1996), isleaf-specific. The Atmyb5 promoter is expressed in developing leaftrichomes, stipules, and epidermal cells on the margins of young rosetteand cauline leaves, and in immature seeds. A leaf promoter identified inmaize by Busk et al. (1997), can also be used.

In some instances, for example when LEC2 or BBM is recombinantlyexpressed, it may be desirable that the transgene is not expressed athigh levels. An example of a promoter which can be used in suchcircumstances is a truncated napin A promoter which retains theseed-specific expression pattern but with a reduced expression level(Tan et al., 2011).

The 5′ non-translated leader sequence can be derived from the promoterselected to express the heterologous gene sequence of the polynucleotideof the present invention, or may be heterologous with respect to thecoding region of the enzyme to be produced, and can be specificallymodified if desired so as to increase translation of mRNA. For a reviewof optimizing expression of transgenes, see Koziel et al. (1996). The 5′non-translated regions can also be obtained from plant viral RNAs(Tobacco mosaic virus, Tobacco etch virus, Maize dwarf mosaic virus,Alfalfa mosaic virus, among others) from suitable eukaryotic genes,plant genes (wheat and maize chlorophyll a/b binding protein geneleader), or from a synthetic gene sequence. The present invention is notlimited to constructs wherein the non-translated region is derived fromthe 5′ non-translated sequence that accompanies the promoter sequence.The leader sequence could also be derived from an unrelated promoter orcoding sequence. Leader sequences useful in context of the presentinvention comprise the maize Hsp70 leader (U.S. Pat. Nos. 5,362,865 and5,859,347), and the TMV omega element.

The termination of transcription is accomplished by a 3′ non-translatedDNA sequence operably linked in the expression vector to thepolynucleotide of interest. The 3′ non-translated region of arecombinant DNA molecule contains a polyadenylation signal thatfunctions in plants to cause the addition of adenylate nucleotides tothe 3′ end of the RNA. The 3′ non-translated region can be obtained fromvarious genes that are expressed in plant cells. The nopaline synthase3′ untranslated region, the 3′ untranslated region from pea smallsubunit Rubisco gene, the 3′ untranslated region from soybean 7S seedstorage protein gene are commonly used in this capacity. The 3′transcribed, non-translated regions containing the polyadenylate signalof Agrobacterium tumor-inducing (Ti) plasmid genes are also suitable.

Recombinant DNA technologies can be used to improve expression of atransformed polynucleotide by manipulating, for example, the efficiencywith which the resultant transcripts are translated by codonoptimisation according to the host cell species or the deletion ofsequences that destabilize transcripts, and the efficiency ofpost-translational modifications.

Transfer Nucleic Acids

Transfer nucleic acids can be used to deliver an exogenouspolynucleotide to a cell and comprise one, preferably two, bordersequences and one or more polynucleotides of interest. The transfernucleic acid may or may not encode a selectable marker. Preferably, thetransfer nucleic acid forms part of a binary vector in a bacterium,where the binary vector further comprises elements which allowreplication of the vector in the bacterium, selection, or maintenance ofbacterial cells containing the binary vector. Upon transfer to aeukaryotic cell, the transfer nucleic acid component of the binaryvector is capable of integration into the genome of the eukaryotic cellor, for transient expression experiments, merely of expression in thecell.

As used herein, the term “extrachromosomal transfer nucleic acid” refersto a nucleic acid molecule that is capable of being transferred from abacterium such as Agrobacterium sp., to a eukaryotic cell such as aplant leaf cell. An extrachromosomal transfer nucleic acid is a geneticelement that is well-known as an element capable of being transferred,with the subsequent integration of a nucleotide sequence containedwithin its borders into the genome of the recipient cell. In thisrespect, a transfer nucleic acid is flanked, typically, by two “border”sequences, although in some instances a single border at one end can beused and the second end of the transferred nucleic acid is generatedrandomly in the transfer process. A polynucleotide of interest istypically positioned between the left border-like sequence and the rightborder-like sequence of a transfer nucleic acid. The polynucleotidecontained within the transfer nucleic acid may be operably linked to avariety of different promoter and terminator regulatory elements thatfacilitate its expression, that is, transcription and/or translation ofthe polynucleotide. Transfer DNAs (T-DNAs) from Agrobacterium sp. suchas Agrobacterium tumefaciens or Agrobacterium rhizogenes, and man madevariants/mutants thereof are probably the best characterized examples oftransfer nucleic acids. Another example is P-DNA (“plant-DNA”) whichcomprises T-DNA border-like sequences from plants.

As used herein, “T-DNA” refers to a T-DNA of an Agrobacteriumtumefaciens Ti plasmid or from an Agrobacterium rhizogenes Ri plasmid,or variants thereof which function for transfer of DNA into plant cells.The T-DNA may comprise an entire T-DNA including both right and leftborder sequences, but need only comprise the minimal sequences requiredin cis for transfer, that is, the right T-DNA border sequence. TheT-DNAs of the invention have inserted into them, anywhere between theright and left border sequences (if present), the polynucleotide ofinterest. The sequences encoding factors required in trans for transferof the T-DNA into a plant cell such as vir genes, may be inserted intothe T-DNA, or may be present on the same replicon as the T-DNA, orpreferably are in trans on a compatible replicon in the Agrobacteriumhost. Such “binary vector systems” are well known in the art. As usedherein, “P-DNA” refers to a transfer nucleic acid isolated from a plantgenome, or man made variants/mutants thereof, and comprises at each end,or at only one end, a T-DNA border-like sequence.

As used herein, a “border” sequence of a transfer nucleic acid can beisolated from a selected organism such as a plant or bacterium, or be aman made variant/mutant thereof. The border sequence promotes andfacilitates the transfer of the polynucleotide to which it is linked andmay facilitate its integration in the recipient cell genome. In anembodiment, a border-sequence is between 10-80 bp in length. Bordersequences from T-DNA from Agrobacterium sp. are well known in the artand include those described in Lacroix et al. (2008).

Whilst traditionally only Agrobacterium sp. have been used to transfergenes to plants cells, there are now a large number of systems whichhave been identified/developed which act in a similar manner toAgrobacterium sp. Several non-Agrobacterium species have recently beengenetically modified to be competent for gene transfer (Chung et al.,2006; Broothaerts et al., 2005). These include Rhizobium sp. NGR234,Sinorhizobium meliloti and Mezorhizobium loti.

Direct transfer of eukaryotic expression plasmids from bacteria toeukaryotic hosts was first achieved several decades ago by the fusion ofmammalian cells and protoplasts of plasmid-carrying Escherichia coli(Schaffner, 1980). Since then, the number of bacteria capable ofdelivering genes into mammalian cells has steadily increased (Weiss,2003), being discovered by four groups independently (Sizemore et al.1995; Courvalin et al., 1995; Powell et al., 1996; Darji et al., 1997).

As used herein, the terms “transfection”, “transformation” andvariations thereof are generally used interchangeably. “Transfected” or“transformed” cells may have been manipulated to introduce thepolynucleotide(s) of interest, or may be progeny cells derivedtherefrom.

Recombinant Cells

The invention also provides a recombinant cell, for example, arecombinant plant cell or fungal cell, which is a host cell transformedwith one or more polynucleotides or vectors defined herein, orcombination thereof. Suitable cells of the invention include any cellthat can be transformed with a polynucleotide or recombinant vector ofthe invention, encoding an RNA, polypeptide or enzyme described herein.The cell is a cell which is thereby capable of being used for producinglipid. The recombinant cell may be a cell in culture, a cell in vitro,or in an organism such as for example, a plant, or in an organ such as,for example, a seed or a leaf. Preferably, the cell is in a plant, morepreferably in the seed of a plant. In one embodiment, the recombinantcell is a non-human cell.

Host cells into which the polynucleotide(s) are introduced can be eitheruntransformed cells or cells that are already transformed with at leastone nucleic acid. Such nucleic acids may be related to lipid synthesis,or unrelated. Host cells of the present invention either can beendogenously (i.e., naturally) capable of producing polypeptide(s)defined herein, in which case the recombinant cell derived therefrom hasan enhanced capability of producing the polypeptide(s), or can becapable of producing said polypeptide(s) only after being transformedwith at least one polynucleotide of the invention. In an embodiment, arecombinant cell of the invention has an enhanced capacity to producenon-polar lipid such as TAG.

Host cells of the present invention can be any cell capable of producingat least one protein described herein, and include fungal (includingyeast), animal such as arthropod, and plant cells such as algal cells.In a preferred embodiment, the plant cell is a seed cell, in particular,a cell in a cotyledon or endosperm of a seed. The host cells may be ofan organism suitable for a fermentation process, such as, for example,Yarrowia lipolytica or other yeasts. In one embodiment, the cell is ananimal cell. The animal cell may be of any type of animal such as, forexample, a non-human animal cell, a non-human vertebrate cell, anon-human mammalian cell, or cells of aquatic animals such as fish orcrustacea, invertebrates, insects, etc. Examples of algal cells usefulas host cells of the present invention include, for example,Chlamydomonas sp. (for example, Chlamydomonas reinhardtii), Dunaliellasp., Haematococcus sp., Chlorella sp., Thraustochytrium sp.,Schizochytrium sp., and Volvox sp.

Transgenic Plants

The invention also provides a plant comprising one or more exogenouspolynucleotides or polypeptides and one or more genetic modifications ofthe invention, a cell of the invention, a vector of the invention, or acombination thereof. The term “plant” when used as a noun refers towhole plants, whilst the term “part thereof” refers to plant organs(e.g., leaves, stems, roots, flowers, fruit), single cells (e.g.,pollen), seed, seed parts such as an embryo, endosperm, scutellum orseed coat, plant tissue such as vascular tissue, plant cells and progenyof the same. As used herein, plant parts comprise plant cells.

As used herein, the terms “in a plant” and “in the plant” in the contextof a modification to the plant means that the modification has occurredin at least one part of the plant, including where the modification hasoccurred throughout the plant, and does not exclude where themodification occurs in only one or more but not all parts of the plant.For example, a tissue-specific promoter is said to be expressed “in aplant”, even though it might be expressed only in certain parts of theplant. Analogously, “a transcription factor polypeptide that increasesthe expression of one or more glycolytic and/or fatty acid biosyntheticgenes in the plant” means that the increased expression occurs in atleast a part of the plant.

As used herein, the term “plant” is used in it broadest sense, includingany organism in the Kingdom Plantae. It also includes red and brownalgae as well as green algae. It includes, but is not limited to, anyspecies of flowering plant, grass, crop or cereal (e.g., oilseed, maize,soybean), fodder or forage, fruit or vegetable plant, herb plant, woodyplant or tree. It is not meant to limit a plant to any particularstructure. It also refers to a unicellular plant (e.g., microalga). Theterm “part thereof” in reference to a plant refers to a plant cell andprogeny of same, a plurality of plant cells, a structure that is presentat any stage of a plant's development, or a plant tissue. Suchstructures include, but are not limited to, leaves, stems, flowers,fruits, nuts, roots, seed, seed coat, embryos. The term “plant tissue”includes differentiated and undifferentiated tissues of plants includingthose present in leaves, stems, flowers, fruits, nuts, roots, seed, forexample, embryonic tissue, endosperm, dermal tissue (e.g., epidermis,periderm), vascular tissue (e.g., xylem, phloem), or ground tissue(comprising parenchyma, collenchyma, and/or sclerenchyma cells), as wellas cells in culture (e.g., single cells, protoplasts, callus, embryos,etc.). Plant tissue may be in planta, in organ culture, tissue culture,or cell culture.

As used herein, the term “vegetative tissue” or “vegetative plant part”is any plant tissue, organ or part other than organs for sexualreproduction of plants. The organs for sexual reproduction of plants arespecifically seed bearing organs, flowers, pollen, fruits and seeds.Vegetative tissues and parts include at least plant leaves, stems(including bolts and tillers but excluding the heads), tubers and roots,but excludes flowers, pollen, seed including the seed coat, embryo andendosperm, fruit including mesocarp tissue, seed-bearing pods andseed-bearing heads. In one embodiment, the vegetative part of the plantis an aerial plant part. In another or further embodiment, thevegetative plant part is a green part such as a leaf or stem.

A “transgenic plant” or variations thereof refers to a plant thatcontains a transgene not found in a wild-type plant of the same species,variety or cultivar. Transgenic plants as defined in the context of thepresent invention include plants and their progeny which have beengenetically modified using recombinant techniques to cause production ofat least one polypeptide defined herein in the desired plant or partthereof. Transgenic plant parts has a corresponding meaning.

The terms “seed” and “grain” are used interchangeably herein. “Grain”refers to mature grain such as harvested grain or grain which is stillon a plant but ready for harvesting, but can also refer to grain afterimbibition or germination, according to the context. Mature graincommonly has a moisture content of less than about 18%. In a preferrdembodiment, the moisture content of the grain is at a level which isgenerally regarded as safe for storage, preferably between 5% and 15%,between 6% and 8%, between 8% and 10%, or between 10% and 15%.“Developing seed” as used herein refers to a seed prior to maturity,typically found in the reproductive structures of the plant afterfertilisation or anthesis, but can also refer to such seeds prior tomaturity which are isolated from a plant. Mature seed commonly has amoisture content of less than about 12%.

As used herein, the term “plant storage organ” refers to a part of aplant specialized to store energy in the form of for example, proteins,carbohydrates, lipid. Examples of plant storage organs are seed, fruit,tuberous roots, and tubers. A preferred plant storage organ of theinvention is seed.

As used herein, the term “phenotypically normal” refers to a geneticallymodified plant or part thereof, for example a transgenic plant, or astorage organ such as a seed, tuber or fruit of the invention not havinga significantly reduced ability to grow and reproduce when compared toan unmodified plant or part thereof. Preferably, the biomass, growthrate, germination rate, storage organ size, seed size and/or the numberof viable seeds produced is not less than 90% of that of a plant lackingsaid genetic modifications or exogenous polynucleotides when grown underidentical conditions. This term does not encompass features of the plantwhich may be different to the wild-type plant but which do not effectthe usefulness of the plant for commercial purposes such as, forexample, a ballerina phenotype of seedling leaves. In an embodiment, thegenetically modified plant or part thereof which is phenotypicallynormal comprises a recombinant polynucleotide encoding a silencingsuppressor operably linked to a plant storage organ specific promoterand has an ability to grow or reproduce which is essentially the same asa corresponding plant or part thereof not comprising saidpolynucleotide.

As used herein, the term “commencement of flowering” or “initiation offlowering” with respect to a plant refers to the time that the firstflower on the plant opens, or the time of onset of anthesis.

As used herein, the term “seed set” with respect to a seed-bearing plantrefers to the time when the first seed of the plant reaches maturity.This is typically observable by the colour of the seed or its moisturecontent, well known in the art.

As used herein, the term “senescence” with respect to a whole plantrefers to the final stage of plant development which follows thecompletion of growth, usually after the plant reaches maximum aerialbiomass or height. Senescence begins when the plant aerial biomassreaches its maximum and begins to decline in amount and generally endswith death of most of the plant tissues. It is during this stage thatthe plant mobilises and recycles cellular components from leaves andother parts which accumulated during growth to other parts to completeits reproductive development. Senescence is a complex, regulated processwhich involves new or increased gene expression of some genes. Often,some plant parts are senescing while other parts of the same plantcontinue to grow. Therefore, with respect to a plant leaf or other greenorgan, the term “senescence” as used herein refers to the time when theamount of chlorophyll in the leaf or organ begins to decrease.Senescence is typically associated with dessication of the leaf ororgan, mostly in the last stage of senescence. Senescence is usuallyobservable by the change in colour of the leaf from green towards yellowand eventually to brown when fully dessicated. It is believed thatcellular senescence underlies plant and organ senescence.

Plants provided by or contemplated for use in the practice of thepresent invention include both monocotyledons and dicotyledons. Inpreferred embodiments, the plants of the present invention are cropplants (for example, cereals and pulses, maize, wheat, potatoes, rice,sorghum, millet, cassava, barley) or legumes such as soybean, beans orpeas. The plants may be grown for production of edible roots, tubers,leaves, stems, flowers or fruit. The plants may be vegetable plantswhose vegetative parts are used as food. The plants of the invention maybe: Acrocomia aculeata (macauba palm), Arabidopsis thaliana, Aracinishypogaea (peanut), Astrocaryum murumuru (murumuru), Astrocaryum vulgare(tucumã), Attalea geraensis (Indaiá-rateiro), Attalea humilis (Americanoil palm), Attalea oleifera (andaiá), Attalea phalerata (uricuri),Attalea speciosa (babassu), Avena sativa (oats), Beta vulgaris (sugarbeet), Brassica sp. such as Brassica carinata, Brassica juncea, Brassicanapobrassica, Brassica napus (canola), Camelina sativa (false flax),Cannabis sativa (hemp), Carthamus tinctorius (safflower), Caryocarbrasiliense (pequi), Cocos nucifera (Coconut), Crambe abyssinica(Abyssinian kale), Cucumis melo (melon), Elaeis guineensis (Africanpalm), Glycine max (soybean), Gossypium hirsutum (cotton), Helianthussp. such as Helianthus annuus (sunflower), Hordeum vulgare (barley),Jatropha curcas (physic nut), Joannesia princeps (arara nut-tree), Lemnasp. (duckweed) such as Lemna aequinoctialis, Lemna disperma, Lemnaecuadoriensis, Lemna gibba (swollen duckweed), Lemna japonica, Lemnaminor, Lemna minuta, Lemna obscura, Lemna paucicostata, Lemnaperpusilla, Lemna lenera, Lemna trisulca, Lemna turionifera, Lemnavaldiviana, Lemna yungensis, Licania rigida (oiticica), Linumusitatissimum (flax), Lupinus angustifolius (lupin), Mauritia flexuosa(buriti palm), Maximiliana maripa (inaja palm), Miscanthus sp. such asMiscanthus x giganteus and Miscanthus sinensis, Nicotiana sp. (tabacco)such as Nicotiana tabacum or Nicotiana benthamiana, Oenocarpus bacaba(bacaba-do-azeite), Oenocarpus bataua (pataul), Oenocarpus distichus(bacaba-de-leque), Oryza sp. (rice) such as Oryza sativa and Oryzaglaberrima, Panicum virgatum (switchgrass), Paraqueiba paraensis (mari),Persea amencana (avocado), Pongamia pinnata (Indian beech), Populustrichocarpa, Ricinus communis (castor), Saccharum sp. (sugarcane),Sesamum indicum (sesame), Solanum tuberosum (potato), Sorghum sp. suchas Sorghum bicolor, Sorghum vulgare, Theobroma grandiforum (cupuassu),Trifolium sp., Trithrinax brasiliensis (Brazilian needle palm), Triticumsp. (wheat) such as Triticum aestivum, Zea mays (corn), alfalfa(Medicago sativa), rye (Secale cerale), sweet potato (Lopmoea batatus),cassava (Manihot esculenta), coffee (Cofea spp.), pineapple (Ananacomosus), citris tree (Citrus spp.), cocoa (Theobroma cacao), tea(Camellia senensis), banana (Musa spp.), avocado (Persea americana), fig(Ficus casica), guava (Psidium guajava), mango (Mangifer indica), olive(Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia intergrifolia) and almond (Prunusamygdalus).

Other preferred plants include C4 grasses such as, in addition to thosementioned above, Andropogon gerardi, Bouteloua curtipendula, B.gracilis, Buchloe dactyloides, Schizachyrium scoparium, Sorghastrumnutans, Sporobolus cryptandrus; C3 grasses such as Elymus canadensis,the legumes Lespedeza capitata and Petalostemum villosum, the forb Asterazureus; and woody plants such as Quercus ellipsoidalis and Q.macrocarpa. Other preferred plants include C3 grasses.

In a preferred embodiment, the plant is an angiosperm.

In an embodiment, the plant is an oilseed plant, preferably an oilseedcrop plant. As used herein, an “oilseed plant” is a plant species usedfor the commercial production of lipid from the seeds of the plant. Theoilseed plant may be, for example, oil-seed rape (such as canola),maize, sunflower, safflower, soybean, sorghum, flax (linseed) or sugarbeet. Furthermore, the oilseed plant may be other Brassicas, cotton,peanut, poppy, rutabaga, mustard, castor bean, sesame, safflower,Jatropha curcas or nut producing plants. The plant may produce highlevels of lipid in its fruit such as olive, oil palm or coconut.Horticultural plants to which the present invention may be applied arelettuce, endive, or vegetable Brassicas including cabbage, broccoli, orcauliflower. The present invention may be applied in tobacco, cucurbits,carrot, strawberry, tomato, or pepper.

In a preferred embodiment, the transgenic plant is homozygous for eachand every gene that has been introduced (transgene) so that its progenydo not segregate for the desired phenotype. The transgenic plant mayalso be heterozygous for the introduced transgene(s), preferablyuniformly heterozygous for the transgene such as for example, in F1progeny which have been grown from hybrid seed. Such plants may provideadvantages such as hybrid vigour, well known in the art.

Transformation of Plants

Transgenic plants can be produced using techniques known in the art,such as those generally described in Slater et al., PlantBiotechnology—The Genetic Manipulation of Plants, Oxford UniversityPress (2003), and Christou and Klee, Handbook of Plant Biotechnology,John Wiley and Sons (2004).

As used herein, the terms “stably transforming”, “stably transformed”and variations thereof refer to the integration of the polynucleotideinto the genome of the cell such that they are transferred to progenycells during cell division without the need for positively selecting fortheir presence. Stable transformants, or progeny thereof, can beidentified by any means known in the art such as Southern blots onchromosomal DNA, or in situ hybridization of genomic DNA, enabling theirselection.

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because DNA can be introduced intocells in whole plant tissues, plant organs, or explants in tissueculture, for either transient expression, or for stable integration ofthe DNA in the plant cell genome. For example, floral-dip (in planta)methods may be used. The use of Agrobacterium-mediated plant integratingvectors to introduce DNA into plant cells is well known in the art. Theregion of DNA to be transferred is defined by the border sequences, andthe intervening DNA (T-DNA) is usually inserted into the plant genome.It is the method of choice because of the facile and defined nature ofthe gene transfer.

Acceleration methods that may be used include for example,microprojectile bombardment and the like. One example of a method fordelivering transforming nucleic acid molecules to plant cells ismicroprojectile bombardment. This method has been reviewed by Yang etal., Particle Bombardment Technology for Gene Transfer, Oxford Press,Oxford, England (1994). Non-biological particles (microprojectiles) thatmay be coated with nucleic acids and delivered into cells, for exampleof immature embryos, by a propelling force. Exemplary particles includethose comprised of tungsten, gold, platinum, and the like.

In another method, plastids can be stably transformed. Methods disclosedfor plastid transformation in higher plants include particle gundelivery of DNA containing a selectable marker and targeting of the DNAto the plastid genome through homologous recombination (U.S. Pat. Nos.5,451,513, 5,545,818, 5,877,402, 5,932,479, and WO 99/05265). Othermethods of cell transformation can also be used and include but are notlimited to the introduction of DNA into plants by direct DNA transferinto pollen, by direct injection of DNA into reproductive organs of aplant, or by direct injection of DNA into the cells of immature embryosfollowed by the rehydration of desiccated embryos.

The regeneration, development, and cultivation of plants from singleplant protoplast transformants or from various transformed explants iswell known in the art (Weissbach et al., In: Methods for Plant MolecularBiology, Academic Press, San Diego, Calif., (1988)). This regenerationand growth process typically includes the steps of selection oftransformed cells, culturing those individualized cells through theusual stages of embryonic development through the rooted plantlet stage.Transgenic embryos and seeds are similarly regenerated. The resultingtransgenic rooted shoots are thereafter planted in an appropriate plantgrowth medium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene is well known in the art. Preferably, the regeneratedplants are self-pollinated to provide homozygous transgenic plants.Otherwise, pollen obtained from the regenerated plants is crossed toseed-grown plants of agronomically important lines. Conversely, pollenfrom plants of these important lines is used to pollinate regeneratedplants. A transgenic plant of the present invention containing a desiredpolynucleotide is cultivated using methods well known to one skilled inthe art.

To confirm the presence of the transgenes in transgenic cells andplants, a polymerase chain reaction (PCR) amplification or Southern blotanalysis can be performed using methods known to those skilled in theart. Expression products of the transgenes can be detected in any of avariety of ways, depending upon the nature of the product, and includeNorthern blot hybridisation, Western blot and enzyme assay. Oncetransgenic plants have been obtained, they may be grown to produce planttissues or parts having the desired phenotype. The plant tissue or plantparts, may be harvested, and/or the seed collected. The seed may serveas a source for growing additional plants with tissues or parts havingthe desired characteristics. Preferably, the vegetative plant parts areharvested at a time when the yield of non-polar lipids are at theirhighest. In one embodiment, the vegetative plant parts are harvestedabout at the time of flowering, or after flowering has initiated.Preferably, the plant parts are harvested at about the time senescencebegins, usually indicated by yellowing and drying of leaves.

Transgenic plants formed using Agrobacterium or other transformationmethods typically contain a single genetic locus on one chromosome. Suchtransgenic plants can be referred to as being hemizygous for the addedgene(s). More preferred is a transgenic plant that is homozygous for theadded gene(s), that is, a transgenic plant that contains two addedgenes, one gene at the same locus on each chromosome of a chromosomepair. A homozygous transgenic plant can be obtained by self-fertilisinga hemizygous transgenic plant, germinating some of the seed produced andanalyzing the resulting plants for the gene of interest.

It is also to be understood that two different transgenic plants thatcontain two independently segregating exogenous genes or loci can alsobe crossed (mated) to produce offspring that contain both sets of genesor loci. Selfing of appropriate F1 progeny can produce plants that arehomozygous for both exogenous genes or loci. Back-crossing to a parentalplant and out-crossing with a non-transgenic plant are alsocontemplated, as is vegetative propagation. Similarly, a transgenicplant can be crossed with a second plant comprising a geneticmodification such as a mutant gene and progeny containing both of thetransgene and the genetic modification identified. Descriptions of otherbreeding methods that are commonly used for different traits and cropscan be found in Fehr, In: Breeding Methods for Cultivar Development,Wilcox J. ed., American Society of Agronomy, Madison Wis. (1987).

Tilling

In one embodiment, TILLING (Targeting Induced Local Lesions IN Genomes)can be used to produce plants in which endogenous genes comprise amutation, for example genes encoding an SDP1 or TGD polypeptide, aplastidial GPAT, plastidial LPAAT, phosphatidic acid phosphatase (PAP),or a combination of two or more thereof. In a first step, introducedmutations such as novel single base pair changes are induced in apopulation of plants by treating seeds (or pollen) with a chemicalmutagen, and then advancing plants to a generation where mutations willbe stably inherited. DNA is extracted, and seeds are stored from allmembers of the population to create a resource that can be accessedrepeatedly over time. For a TILLING assay, heteroduplex methods usingspecific endonucleases can be used to detect single nucleotidepolymorphisms (SNPs). Alternatively, Next Generation Sequencing of DNAfrom pools of mutagenised plants can be used to identify mutants in thegene of choice. Typically, a mutation frequency of one mutant per 1000plants in the mutagenised population is achieved. Using this approach,many thousands of plants can be screened to identify any individual witha single base change as well as small insertions or deletions (1-30 bp)in any gene or specific region of the genome. TILLING is furtherdescribed in Slade and Knauf (2005), and Henikoff et al. (2004).

In addition to allowing efficient detection of mutations,high-throughput TILLING technology is ideal for the detection of naturalpolymorphisms. Therefore, interrogating an unknown homologous DNA byheteroduplexing to a known sequence reveals the number and position ofpolymorphic sites. Both nucleotide changes and small insertions anddeletions are identified, including at least some repeat numberpolymorphisms. This has been called Ecotilling (Comai et al., 2004).

Genome Editing Using Site-Specific Nucleases

Genome editing uses engineered nucleases such as RNA guided DNAendonucleases or nucleases composed of sequence specific DNA bindingdomains fused to a non-specific DNA cleavage module. These engineerednucleases enable efficient and precise genetic modifications by inducingtargeted DNA double stranded breaks that stimulate the cell's endogenouscellular DNA repair mechanisms to repair the induced break. Suchmechanisms include, for example, error prone non-homologous end joining(NHEJ) and homology directed repair (HDR).

In the presence of donor plasmid with extended homology arms, HDR canlead to the introduction of single or multiple transgenes to correct orreplace existing genes. In the absence of donor plasmid, NHEJ-mediatedrepair yields small insertion or deletion mutations of the target thatcause gene disruption.

Engineered nucleases useful in the methods of the present inventioninclude zinc finger nucleases (ZFNs), transcription activator-like (TAL)effector nucleases (TALEN) and CRISPR/Cas9 type nucleases.

Typically nuclease encoded genes are delivered into cells by plasmidDNA, viral vectors or in vitro transcribed mRNA.

A zinc finger nuclease (ZFN) comprises a DNA-binding domain and aDNA-cleavage domain, wherein the DNA binding domain is comprised of atleast one zinc finger and is operatively linked to a DNA-cleavagedomain. The zinc finger DNA-binding domain is at the N-terminus of theprotein and the DNA-cleavage domain is located at the C-terminus of saidprotein.

A ZFN must have at least one zinc finger. In a preferred embodiment, aZFN would have at least three zinc fingers in order to have sufficientspecificity to be useful for targeted genetic recombination in a hostcell or organism. Typically, a ZFN having more than three zinc fingerswould have progressively greater specificity with each additional zincfinger.

The zinc finger domain can be derived from any class or type of zincfinger. In a particular embodiment, the zinc finger domain comprises theCis₂His₂ type of zinc finger that is very generally represented, forexample, by the zinc finger transcription factors TFIIIA or Sp1. In apreferred embodiment, the zinc finger domain comprises three Cis₂His2type zinc fingers. The DNA recognition and/or the binding specificity ofa ZFN can be altered in order to accomplish targeted geneticrecombination at any chosen site in cellular DNA. Such modification canbe accomplished using known molecular biology and/or chemical synthesistechniques. (see, for example, Bibikova et al., 2002).

The ZFN DNA-cleavage domain is derived from a class of non-specific DNAcleavage domains, for example the DNA-cleavage domain of a Type IIrestriction enzyme such as FokI (Kim et al., 1996). Other usefulendonucleases may include, for example, HhaI, HindIII, Nod, BbvCI,EcoRI, BglI, and AwlI.

A transcription activator-like (TAL) effector nuclease (TALEN) comprisesa TAL effector DNA binding domain and an endonuclease domain.

TAL effectors are proteins of plant pathogenic bacteria that areinjected by the pathogen into the plant cell, where they travel to thenucleus and function as transcription factors to turn on specific plantgenes. The primary amino acid sequence of a TAL effector dictates thenucleotide sequence to which it binds. Thus, target sites can bepredicted for TAL effectors, and TAL effectors can be engineered andgenerated for the purpose of binding to particular nucleotide sequences.

Fused to the TAL effector-encoding nucleic acid sequences are sequencesencoding a nuclease or a portion of a nuclease, typically a nonspecificcleavage domain from a type II restriction endonuclease such as FokI(Kim et al., 1996). Other useful endonucleases may include, for example,HhaI, HindIII, Nod, BbvCI, EcoRI, BglI, and AlwI. The fact that someendonucleases (e.g., FokI) only function as dimers can be capitalizedupon to enhance the target specificity of the TAL effector. For example,in some cases each FokI monomer can be fused to a TAL effector sequencethat recognizes a different DNA target sequence, and only when the tworecognition sites are in close proximity do the inactive monomers cometogether to create a functional enzyme. By requiring DNA binding toactivate the nuclease, a highly site-specific restriction enzyme can becreated.

A sequence-specific TALEN can recognize a particular sequence within apreselected target nucleotide sequence present in a cell. Thus, in someembodiments, a target nucleotide sequence can be scanned for nucleaserecognition sites, and a particular nuclease can be selected based onthe target sequence. In other cases, a TALEN can be engineered to targeta particular cellular sequence.

Genome Editing Using Programmable RNA-Guided DNA Endonucleases

Distinct from the site-specific nucleases described above, the clusteredregulatory interspaced short palindromic repeats (CRISPR)/Cas systemprovides an alternative to ZFNs and TALENs for inducing targeted geneticalterations, via RNA-guided DNA cleavage.

CRISPR systems rely on CRISPR RNA (crRNA) and transactivating chimericRNA (tracrRNA) for sequence-specific cleavage of DNA. Three types ofCRISPR/Cas systems exist: in type II systems, Cas9 serves as anRNA-guided DNA endonuclease that cleaves DNA upon crRNA-tracrRNA targetrecognition. CRISPR RNA base pairs with tracrRNA to form a two-RNAstructure that guides the Cas9 endonuclease to complementary DNA sitesfor cleavage.

The CRISPR system can be portable to plant cells by co-delivery ofplasmids expressing the Cas endonuclease and the necessary crRNAcomponents. The Cas endonuclease may be converted into a nickase toprovide additional control over the mechanism of DNA repair (Cong etal., 2013).

CRISPRs are typically short partially palindromic sequences of 24-40 bpcontaining inner and terminal inverted repeats of up to 11 bp. Althoughisolated elements have been detected, they are generally arranged inclusters (up to about 20 or more per genome) of repeated units spaced byunique intervening 20-58 bp sequences. CRISPRs are generally homogenouswithin a given genome with most of them being identical. However, thereare examples of heterogeneity in, for example, the Archaea (Mojica etal., 2000).

Plant Biomass

An increase in the total lipid content of plant biomass equates togreater energy content, making its use as a feed or forage or in theproduction of biofuel more economical.

The main components of naturally occurring plant biomass arecarbohydrates (approximately 75%, dry weight) and lignin (approximately25%), which can vary with plant type. The carbohydrates are mainlycellulose or hemicellulose fibers, which impart strength to the plantstructure, and lignin, which holds the fibers together. Plant biomasstypically has a low energy density as a result of both its physical formand moisture content. This also makes it inconvenient and inefficientfor storage and transport without some kind of pre-processing. There area range of processes available to convert it into a more convenient formincluding: 1) physical pre-processing (for example, grinding) or 2)conversion by thermal (for example, combustion, gasification, pyrolysis)or chemical (for example, anaerobic digestion, fermentation, composting,transesterification) processes. In this way, the biomass is convertedinto what can be described as a biomass fuel.

Combustion

Combustion is the process by which flammable materials are allowed toburn in the presence of air or oxygen with the release of heat. Thebasic process is oxidation. Combustion is the simplest method by whichbiomass can be used for energy, and has been used to provide heat. Thisheat can itself be used in a number of ways: 1) space heating, 2) water(or other fluid) heating for central or district heating or processheat, 3) steam raising for electricity generation or motive force. Whenthe flammable fuel material is a form of biomass the oxidation is ofpredominantly the carbon (C) and hydrogen (H) in the cellulose,hemicellulose, lignin, and other molecules present to form carbondioxide (CO₂) and water (H₂O). The plants of the invention provideimproved fuel for combustion by virtue of the increased lipid content.

Gasification

Gasification is a partial oxidation process whereby a carbon source suchas plant biomass, is broken down into carbon monoxide (CO) and hydrogen(H₂), plus carbon dioxide (CO₂) and possibly hydrocarbon molecules suchas methane (CH₄). If the gasification takes place at a relatively lowtemperature, such as 700° C. to 1000° C., the product gas will have arelatively high level of hydrocarbons compared to high temperaturegasification. As a result it may be used directly, to be burned for heator electricity generation via a steam turbine or, with suitable gasclean up, to run an internal combustion engine for electricitygeneration. The combustion chamber for a simple boiler may be closecoupled with the gasifier, or the producer gas may be cleaned of longerchain hydrocarbons (tars), transported, stored and burned remotely. Agasification system may be closely integrated with a combined cycle gasturbine for electricity generation (IGCC—integrated gasificationcombined cycle). Higher temperature gasification (1200° C. to 1600° C.)leads to few hydrocarbons in the product gas, and a higher proportion ofCO and H₂. This is known as synthesis gas (syngas or biosyngas) as itcan be used to synthesize longer chain hydrocarbons using techniquessuch as Fischer-Tropsch (FT) synthesis. If the ratio of H₂ to CO iscorrect (2:1) FT synthesis can be used to convert syngas into highquality synthetic diesel biofuel which is compatible with conventionalfossil diesel and diesel engines.

Pyrolysis

As used herein, the term “pyrolysis” means a process that uses slowheating in the absence of oxygen to produce gaseous, oil and charproducts from biomass. Pyrolysis is a thermal or thermo-chemicalconversion of lipid-based, particularly triglyceride-based, materials.The products of pyrolysis include gas, liquid and a sold char, with theproportions of each depending upon the parameters of the process. Lowertemperatures (around 400° C.) tend to produce more solid char (slowpyrolysis), whereas somewhat higher temperatures (around 500° C.)produce a much higher proportion of liquid (bio-oil), provided thevapour residence time is kept down to around Is or less. Temperatures ofabout 275° C. to about 375° C. can be used to produce liquid bio-oilhaving a higher proportion of longer chain hydrocarbons. Pyrolysisinvolves direct thermal cracking of the lipids or a combination ofthermal and catalytic cracking. At temperatures of about 400-500° C.,cracking occurs, producing short chain hydrocarbons such as alkanes,alkenes, alkadienes, aromatics, olefins and carboxylic acid, as well ascarbon monoxide and carbon dioxide.

Four main catalyst types can be used including transition metalcatalysts, molecular sieve type catalysts, activated alumina and sodiumcarbonate (Maher et al., 2007). Examples are given in U.S. Pat. No.4,102,938. Alumina (Al₂O₃) activated by acid is an effective catalyst(U.S. Pat. No. 5,233,109). Molecular sieve catalysts are porous, highlycrystalline structures that exhibit size selectivity, so that moleculesof only certain sizes can pass through. These include zeolite catalystssuch as ZSM-5 or HZSM-5 which are crystalline materials comprising AlO₄and SiO₄ and other silica-alumina catalysts. The activity andselectivity of these catalysts depends on the acidity, pore size andpore shape, and typically operate at 300-500° C. Transition metalcatalysts are described for example in U.S. Pat. No. 4,992,605. Sodiumcarbonate catalyst has been used in the pyrolysis of oils (Dandik andAksoy, 1998).

As used herein, “hydrothermal processing”, “HTP”, also referred to as“thermal depolymerisation” is a form of pyrolysis which reacts theplant-derived matter, specifically the carbon-containing material in theplant-derived matter, with hydrogen to produce a bio-oil productcomprised predominantly of paraffinic hydrocarbons along with othergases and solids. A significant advantage of HTP is that the vegetativeplant material does not need to be dried before forming the compositionfor the conversion reaction, although the vegetative plant material canbe dried beforehand to aid in transport or storage of the biomass. Thebiomass can be used directly as harvested from the field. The reactor isany vessel which can withstand the high temperature and pressure usedand is resistant to corrosion. The solvent used in the HTP includeswater or is entirely water, or may include some hydrocarbon compounds inthe form of an oil. Generally, the solvent in HTP lacks added alcohols.The conversion reaction may occur in an oxidative, reductive or inertenvironment. “Oxidative” as used herein means in the presence of air,“reductive” means in the presence of a reducing agent, typicallyhydrogen gas or methane, for example 10-15% H₂ with the remainder of thegas being N₂, and “inert” means in the presence of an inert gas such asnitrogen or argon. The conversion reaction is preferably carried outunder reductive conditions. The carbon-containing materials that areconverted include cellulose, hemi-cellulose, lignin and proteins as wellas lipids. The process uses a conversion temperature of between 270° C.and 400° C. and a pressure of between 70 and 350 bar, typically 300° C.to 350° C. and a pressure between 100-170 bar. As a result of theprocess, organic vapours, pyrolysis gases and charcoal are produced. Theorganic vapours are condensed to produce the bio-oil. Recovery of thebio-oil may be achieved by cooling the reactor and reducing the pressureto atmospheric pressure, which allows bio-oil (organic) and water phasesto develop and the bio-oil to be removed from the reactor.

The yield of the recovered bio-oil is calculated as a percentage of thedry weight of the input biomass on a dry weight basis. It is calculatedaccording to the formula: weight of bio-oil×100/dry weight of thevegetative plant parts. The weight of the bio-oil does not include theweight of any water or solids which may be present in a bio-oil mixture,which are readily removed by filtration or other known methods.

The bio-oil may then be separated into fractions by fractionaldistillation, with or without additional refining processes. Typically,the fractions that condense at these temperatures are termed: about 370°C., fuel oil; about 300° C., diesel oil; about 200° C., kerosene; about150° C., gasoline (petrol). Heavier fractions may be cracked intolighter, more desirable fractions, well known in the art. Diesel fueltypically is comprised of C13-C22 hydrocarbon compounds.

As used herein, “petroleum diesel” (petrodiesel) means a diesel fuelmade from fossil fuel and which falls under the specifications outlinedby ASTM D975 in the United States and EN 590 in Europe. The term“renewable diesel” as used herein means a diesel fuel derived fromrecently living biomass (not fossil fuel) that meets the standards ofASTM D975 and are not mono-alkyl esters. Typical features of renewablediesel are: cetane number of 75-90, energy density of about 44 MJ/kg,density of about 0.78 g/ml, energy content of about 123 K BTU/gal,sulphur levels less than 10 ppm, cloud point below 0° C.

Transesterification

“Transesterification” as used herein is the conversion of lipids,principally triacylglycerols, into fatty acid methyl esters or ethylesters by reaction with short chain alcohols such as methanol orethanol, in the presence of a catalyst such as alkali or acid. Methanolis used more commonly due to low cost and availability, but ethanol,propanol or butanol or mixtures of the alcohols can also be used. Thecatalysts may be homogeneous catalysts, heterogeneous catalysts orenzymatic catalysts. Homogeneous catalysts include ferric sulphatefollowed by KOH. Heterogeneous catalysts include CaO, K₃PO₄, andWO₃/ZrO₂. Enzymatic catalysts include Novozyme 435 produced from Candidaantarctica.

Transesterification can be carried out on extracted oil, or preferablydirectly in situ in the vegetative plant material. The vegetative plantparts may be dried and milled prior to being used to prepare thecomposition for the conversion reaction, but does not need to be. Theadvantage of direct conversion to fatty acid esters, preferably FAME, isthat the conversion can use lower temperatures and pressures and stillprovide good yields of the product, for example, comprising at least 50%FAME by weight. The yield of recovered bio-oil by transesterification iscalculated as for the HTP process.

Production of Non-Polar Lipids

Techniques that are routinely practiced in the art can be used toextract, process, purify and analyze the lipids such as the TAG producedby cells, organisms or parts thereof of the instant invention. Suchtechniques are described and explained throughout the literature insources such as, Fereidoon Shahidi, Current Protocols in Food AnalyticalChemistry, John Wiley & Sons, Inc. (2001) D1.1.1-D1.1.11, and Perez-Vichet al. (1998).

Production of Oil from Vegetative Plant Parts or Seed

Typically, plant seeds are cooked, pressed, and/or extracted to producecrude seedoil, which is then degummed, refined, bleached, anddeodorized. Generally, techniques for crushing seed are known in theart. For example, oilseeds can be tempered by spraying them with waterto raise the moisture content to, for example, 8.5%, and flaked using asmooth roller with a gap setting of 0.23 to 0.27 mm. Depending on thetype of seed, water may not be added prior to crushing. Application ofheat deactivates enzymes, facilitates further cell rupturing, coalescesthe lipid droplets, and agglomerates protein particles, all of whichfacilitate the extraction process.

In an embodiment, the majority of the seedoil is released by passagethrough a screw press. Cakes expelled from the screw press are thensolvent extracted for example, with hexane, using a heat traced column.Alternatively, crude seedoil produced by the pressing operation can bepassed through a settling tank with a slotted wire drainage top toremove the solids that are expressed with the seedoil during thepressing operation. The clarified seedoil can be passed through a plateand frame filter to remove any remaining fine solid particles. Ifdesired, the seedoil recovered from the extraction process can becombined with the clarified seedoil to produce a blended crude seedoil.

Once the solvent is stripped from the crude seedoil, the pressed andextracted portions are combined and subjected to normal lipid processingprocedures (i.e., degumming, caustic refining, bleaching, anddeodorization).

Extraction of the lipid from vegetative plant parts of the inventionuses analogous methods to those known in the art for seedoil extraction.One way is physical extraction, which often does not use solventextraction. Expeller pressed extraction is a common type, as are thescrew press and ram press extraction methods. Mechanical extraction istypically less efficient than solvent extraction where an organicsolvent (e.g., hexane) is mixed with at least the plant biomass,preferably after the biomass is dried and ground. The solvent dissolvesthe lipid in the biomass, which solution is then separated from thebiomass by mechanical action (e.g., with the pressing processes above).This separation step can also be performed by filtration (e.g., with afilter press or similar device) or centrifugation etc. The organicsolvent can then be separated from the non-polar lipid (e.g., bydistillation). This second separation step yields non-polar lipid fromthe plant and can yield a re-usable solvent if one employs conventionalvapor recovery. In an embodiment, the oil and/or protein content of theplant part or seed is analysed by near-infrared reflectance spectroscopyas described in Hom et al. (2007) prior to extraction.

If the vegetative plant parts are not to be used immediately to extractthe lipid it is preferably processed to ensure the lipid content isminimized as much as possible (see, for example, Christie, 1993), suchas by drying the vegetative plant parts.

Degumming

Degumming is an early step in the refining of oils and its primarypurpose is the removal of most of the phospholipids from the oil, whichmay be present as approximately 1-2% of the total extracted lipid.Addition of ˜2% of water, typically containing phosphoric acid, at70-80° C. to the crude oil results in the separation of most of thephospholipids accompanied by trace metals and pigments. The insolublematerial that is removed is mainly a mixture of phospholipids andtriacylglycerols and is also known as lecithin. Degumming can beperformed by addition of concentrated phosphoric acid to the crudeseedoil to convert non-hydratable phosphatides to a hydratable form, andto chelate minor metals that are present. Gum is separated from theseedoil by centrifugation. The seedoil can be refined by addition of asufficient amount of a sodium hydroxide solution to titrate all of thefatty acids and removing the soaps thus formed.

Alkali Refining

Alkali refining is one of the refining processes for treating crude oil,sometimes also referred to as neutralization. It usually followsdegumming and precedes bleaching. Following degumming, the seedoil cantreated by the addition of a sufficient amount of an alkali solution totitrate all of the fatty acids and phosphoric acids, and removing thesoaps thus formed. Suitable alkaline materials include sodium hydroxide,potassium hydroxide, sodium carbonate, lithium hydroxide, calciumhydroxide, calcium carbonate and ammonium hydroxide. This process istypically carried out at room temperature and removes the free fattyacid fraction. Soap is removed by centrifugation or by extraction into asolvent for the soap, and the neutralised oil is washed with water. Ifrequired, any excess alkali in the oil may be neutralized with asuitable acid such as hydrochloric acid or sulphuric acid.

Bleaching

Bleaching is a refining process in which oils are heated at 90-120° C.for 10-30 minutes in the presence of a bleaching earth (0.2-2.0%) and inthe absence of oxygen by operating with nitrogen or steam or in avacuum. This step in oil processing is designed to remove unwantedpigments (carotenoids, chlorophyll, gossypol etc), and the process alsoremoves oxidation products, trace metals, sulphur compounds and tracesof soap.

Deodorization

Deodorization is a treatment of oils and fats at a high temperature(200-260° C.) and low pressure (0.1-1 mm Hg). This is typically achievedby introducing steam into the seedoil at a rate of about 0.1ml/minute/100 ml of seedoil. Deodorization can be performed by heatingthe seedoil to 260° C. under vacuum, and slowly introducing steam intothe seedoil at a rate of about 0.1 ml/minute/100 ml of seedoil. Afterabout 30 minutes of sparging, the seedoil is allowed to cool undervacuum. The seedoil is typically transferred to a glass container andflushed with argon before being stored under refrigeration. If theamount of seedoil is limited, the seedoil can be placed under vacuum forexample, in a Parr reactor and heated to 260° C. for the same length oftime that it would have been deodorized. This treatment improves thecolour of the seedoil and removes a majority of the volatile substancesor odorous compounds including any remaining free fatty acids,monoacylglycerols and oxidation products.

Winterisation

Winterization is a process sometimes used in commercial production ofoils for the separation of oils and fats into solid (stearin) and liquid(olein) fractions by crystallization at sub-ambient temperatures. It wasapplied originally to cottonseed oil to produce a solid-free product. Itis typically used to decrease the saturated fatty acid content of oils.

Algae for the Production of Lipids

Algae can produce 10 to 100 times as much mass as terrestrial plants ina year and can be cultured in open-ponds (such as raceway-type ponds andlakes) or in photobioreactors. The most common oil-producing algae cangenerally include the diatoms (bacillariophytes), green algae(chlorophytes), blue-green algae (cyanophytes), and golden-brown algae(chrysophytes). In addition a fifth group known as haptophytes may beused. Groups include brown algae and heterokonts. Specific non-limitingexamples algae include the Classes: Chlorophyceae, Eustigmatophyceae,Prymnesiophyceae, Bacillariophyceae. Bacillariophytes capable of oilproduction include the genera Amphipleura, Amphora, Chaetoceros,Cyclotella, Cymbella, Fragilaria, Hantzschia, Navicula, Nitzschia,Phaeodactylum, and Thalassiosira. Specific non-limiting examples ofchlorophytes capable of oil production include Ankistrodesmus,Botryococcus, Chlorella, Chlorococcum, Dunaliella, Monoraphidium,Oocystis, Scenedesmus, and Tetraselmis. In one aspect, the chlorophytescan be Chlorella or Dunaliella. Specific non-limiting examples ofcyanophytes capable of oil production include Oscillatoria andSynechococcus. A specific example of chrysophytes capable of oilproduction includes Boekelovia. Specific non-limiting examples ofhaptophytes include Isochysis and Pleurochysis.

Specific algae useful in the present invention include, for example,Chlamydomonas sp. such as Chlamydomonas reinhardtii, Dunaliella sp. suchas Dunaliella salina, Dunaliella tertiolecta, D. acidophila, D.Lateralis. D. martima. D. parva, D. polmorpha, D. primolecta, D.pseudosalina, D. quartolecta. D. viridis, Haematococcus sp., Chlorellasp. such as Chlorella vulgaris, Chlorella sorokiniana or Chlorellaprotothecoides, Thraustochytrium sp., Schizochytrium sp., Volvox sp,Nannochloropsis sp., Botryococcus braunii which can contain over 60 wt %lipid, Phaeodactylum tricornutum, Thalassiosira pseudonana, Isochrysissp., Pavlova sp., Chlorococcum sp, Ellipsoidion sp., Neochloris sp.,Scenedesmus sp.

Algae of the invention can be harvested using microscreens, bycentrifugation, by flocculation (using for example, chitosan, alum andferric chloride) and by froth flotation. Interrupting the carbon dioxidesupply can cause algae to flocculate on its own, which is called“autoflocculation”. In froth flotation, the cultivator aerates the waterinto a froth, and then skims the algae from the top. Ultrasound andother harvesting methods are currently under development.

Lipid may be extracted from the algae by mechanical crushing. When algalmass is dried it retains its lipid content, which can then be “pressed”out with an oil press. Osmotic shock may also be used to releasecellular components such as lipid from algae, and ultrasonic extractioncan accelerate extraction processes. Chemical solvents (for example,hexane, benzene, petroleum ether) are often used in the extraction oflipids from algae. Enzymatic extraction ting enzymes to degrade the cellwalls may also be used to extract lipids from algae. Supercritical CO₂can also be used as a solvent. In this method, CO₂ is liquefied underpressure and heated to the point that it becomes supercritical (havingproperties of both a liquid and a gas), allowing it to act as a solvent.

As used herein, an “oleaginous organism” is one which accumulates atleast 20% of its dry weight as triacylglycerols. As used herein, “yeast”includes Saccharomyces spp., Saccharomyces cerevisiae, Saccharomycescarlbergensis, Candida spp., Kluveromyces spp., Pichia spp., Hansenulaspp., Trichoderma spp., Lipomyces starkey, and Yarrowia lipolytica.Preferred yeast include Yarrowia lipolytica or other oleaginous yeastsand strains of the Saccharomyces spp.

Uses of Plant Lipids

The lipids produced by the methods described have a variety of uses. Insome embodiments, the lipids are used as food oils. In otherembodiments, the lipids are refined and used as lubricants or for otherindustrial uses such as the synthesis of plastics. In some preferredembodiments, the lipids are refined to produce biodiesel. Biodiesel canbe made from oils derived from the plants, algae and fungi of theinvention. Use of plant triacylglycerols for the production of biofuelis reviewed in Durrett et al. (2008). The resulting fuel is commonlyreferred to as biodiesel and has a dynamic viscosity range from 1.9 to6.0 mm² s⁻¹ (ASTM D6751). Bioalcohol may produced from the fermentationof sugars or the biomass other than the lipid left over after lipidextraction. General methods for the production of biofuel can be foundin, for example, Maher and Bressler (2007), Greenwell et al. (2010),Karmakar et al. (2010), Alonso et al. (2010), Liu et al. (2010a). Gongand Jiang (2011), Endalew et al. (2011) and Semwal et al. (2011).

The present invention provides methods for increasing oil content invegetative tissues. Plants of the present invention have increasedenergy content of leaves and/or stems such that the whole above-groundplant parts may be harvested and used to produce biofuel. Furthermore,the level of oleic acid is increased significantly while thepolyunsaturated fatty acid alpha linolenic acid (ALA) was reduced. Theplants, algae and fungi of the present invention thereby reduce theproduction costs of biofuel.

Biodiesel

The production of biodiesel, or alkyl esters, is well known. There arethree basic routes to ester production from lipids: 1) Base catalysedtransesterification of the lipid with alcohol; 2) Direct acid catalysedesterification of the lipid with methanol; and 3) Conversion of thelipid to fatty acids, and then to alkyl esters with acid catalysis. Anymethod for preparing fatty acid alkyl esters and glyceryl ethers (inwhich one, two or three of the hydroxy groups on glycerol areetherified) can be used. For example, fatty acids can be prepared, forexample, by hydrolyzing or saponifying TAG with acid or base catalysts,respectively, or using an enzyme such as a lipase or an esterase. Fattyacid alkyl esters can be prepared by reacting a fatty acid with analcohol in the presence of an acid catalyst. Fatty acid alkyl esters canalso be prepared by reacting TAG with an alcohol in the presence of anacid or base catalyst. Glycerol ethers can be prepared, for example, byreacting glycerol with an alkyl halide in the presence of base, or withan olefin or alcohol in the presence of an acid catalyst. The alkylesters can be directly blended with diesel fuel, or washed with water orother aqueous solutions to remove various impurities, including thecatalysts, before blending.

Aviation Fuel

For improved performance of biofuels, thermal and catalytic chemicalbond-breaking (cracking) technologies have been developed that enableconverting bio-oils into bio-based alternatives to petroleum-deriveddiesel fuel and other fuels, such as jet fuel.

The use of medium chain fatty acid source, such produced by arecombinant eukaryotic cell of the invention, a transgenic non-humanorganism or a part thereof of the invention, a transgenic plant or partthereof of the invention, a seed of of the invention, or a transgeniccell or transgenic plant or part thereof of the invention, precludes theneed for high-energy fatty acid chain cracking to achieve the shortermolecules needed for jet fuels and other fuels with low-temperature flowrequirements. This method comprises cleaving one or more medium chainfatty acid groups from the glycerides to form glycerol and one or morefree fatty acids. In addition, the method comprises separating the oneor more medium chain fatty acids from the glycerol, and decarboxylatingthe one or more medium chain fatty acids to form one or morehydrocarbons for the production of the jet fuel.

Feedstufs

The present invention includes compositions which can be used asfeedstuffs. For purposes of the present invention, “feedstuffs” includeany food or preparation for human or animal consumption and which servesto nourish or build up tissues or supply energy, and/or to maintain,restore or support adequate nutritional status or metabolic function.Feedstuffs of the invention include nutritional compositions for babiesand/or young children.

Feedstuffs of the invention comprise for example, a cell of theinvention, a plant of the invention, the plant part of the invention,the seed of the invention, an extract of the invention, the product of amethod of the invention or a composition along with a suitablecarrier(s). The term “carrier” is used in its broadest sense toencompass any component which may or may not have nutritional value. Asthe person skilled in the art will appreciate, the carrier must besuitable for use (or used in a sufficiently low concentration) in afeedstuff, such that it does not have deleterious effect on an organismwhich consumes the feedstuff.

The feedstuff of the present invention comprises a lipid produceddirectly or indirectly by use of the methods, cells or organismsdisclosed herein. The composition may either be in a solid or liquidform. Additionally, the composition may include edible macronutrients,vitamins, and/or minerals in amounts desired for a particular use. Theamounts of these ingredients will vary depending on whether thecomposition is intended for use with normal individuals or for use withindividuals having specialized needs such as individuals suffering frommetabolic disorders and the like.

Examples of suitable carriers with nutritional value include, but arenot limited to, macronutrients such as edible fats, carbohydrates andproteins. Examples of such edible fats include, but are not limited to,coconut oil, borage oil, fungal oil, black current oil, soy oil, andmono- and di-glycerides. Examples of such carbohydrates include, but arenot limited to, glucose, edible lactose, and hydrolyzed starch.Additionally, examples of proteins which may be utilized in thenutritional composition of the invention include, but are not limitedto, soy proteins, electrodialysed whey, electrodialysed skim milk, milkwhey, or the hydrolysates of these proteins.

With respect to vitamins and minerals, the following may be added to thefeedstuff compositions of the present invention, calcium, phosphorus,potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc,selenium, iodine, and vitamins A, E, D, C, and the B complex. Other suchvitamins and minerals may also be added.

A feedstuff composition of the present invention may also be added tofood even when supplementation of the diet is not required. For example,the composition may be added to food of any type, including, but notlimited to, margarine, butter, cheeses, milk, yogurt, chocolate, candy,snacks, salad oils, cooking oils, cooking fats, meats, fish andbeverages.

Additionally, lipid produced in accordance with the present invention orhost cells transformed to contain and express the subject genes may alsobe used as animal food supplements to alter an animal's tissue or milkfatty acid composition to one more desirable for human or animalconsumption. Examples of such animals include sheep, cattle, horses andthe like. Furthermore, feedstuffs of the invention can be used inaquaculture to increase the levels of fatty acids in fish for human oranimal consumption.

Preferred feedstuffs of the invention are the plants, seed and otherplant parts such as leaves, fruits and stems which may be used directlyas food or feed for humans or other animals. For example, animals maygraze directly on such plants grown in the field, or be fed moremeasured amounts in controlled feeding. The invention includes the useof such plants and plant parts as feed for increasing thepolyunsaturated fatty acid levels in humans and other animals.

For consumption by non-human animals the feedstuff may be in anysuitable form for such as, but not limited to, silage, hay or pasturegrowing in a field. In an embodiment, the feedstuff for non-humanconsumption is a leguminous plant, or part thereof, which is a member ofthe family Fabaceae family (or Leguminosae) such as alfalfa, clover,peas, luceme, beans, lentils, lupins, mesquite, carob, soybeans, andpeanuts.

Compositions

The present invention also encompasses compositions, particularlypharmaceutical compositions, comprising one or more lipids producedusing the methods of the invention.

A pharmaceutical composition may comprise one or more of the lipids, incombination with a standard, well-known, non-toxicpharmaceutically-acceptable carrier, adjuvant or vehicle such asphosphate-buffered saline, water, ethanol, polyols, vegetable oils, awetting agent, or an emulsion such as a water/oil emulsion. Thecomposition may be in either a liquid or solid form. For example, thecomposition may be in the form of a tablet, capsule, ingestible liquid,powder, topical ointment or cream. Proper fluidity can be maintained forexample, by the maintenance of the required particle size in the case ofdispersions and by the use of surfactants. It may also be desirable toinclude isotonic agents for example, sugars, sodium chloride, and thelike. Besides such inert diluents, the composition can also includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening agents, flavoring agents and perfuming agents.

A typical dosage of a particular fatty acid is from 0.1 mg to 20 g,taken from one to five times per day (up to 100 g daily) and ispreferably in the range of from about 10 mg to about 1, 2, 5, or 10 gdaily (taken in one or multiple doses). As known in the art, a minimumof about 300 mg/day of fatty acid, especially polyunsaturated fattyacid, is desirable. However, it will be appreciated that any amount offatty acid will be beneficial to the subject.

Possible routes of administration of the pharmaceutical compositions ofthe present invention include for example, enteral and parenteral. Forexample, a liquid preparation may be administered orally. Additionally,a homogenous mixture can be completely dispersed in water, admixed understerile conditions with physiologically acceptable diluents,preservatives, buffers or propellants to form a spray or inhalant.

The dosage of the composition to be administered to the subject may bedetermined by one of ordinary skill in the art and depends upon variousfactors such as weight, age, overall health, past history, immunestatus, etc., of the subject.

Additionally, the compositions of the present invention may be utilizedfor cosmetic purposes. The compositions may be added to pre-existingcosmetic compositions, such that a mixture is formed, or a fatty acidproduced according to the invention may be used as the sole “active”ingredient in a cosmetic composition.

Polypeptides

The terms “polypeptide” and “protein” are generally used interchangeablyherein.

A polypeptide or class of polypeptides may be defined by the extent ofidentity (% identity) of its amino acid sequence to a reference aminoacid sequence, or by having a greater % identity to one reference aminoacid sequence than to another. The % identity of a polypeptide to areference amino acid sequence is typically determined by GAP analysis(Needleman and Wunsch, 1970; GCG program) with parameters of a gapcreation penalty=5, and a gap extension penalty=0.3. The query sequenceis at least 100 amino acids in length and the GAP analysis aligns thetwo sequences over a region of at least 100 amino acids. Even morepreferably, the query sequence is at least 250 amino acids in length andthe GAP analysis aligns the two sequences over a region of at least 250amino acids. Even more preferably, the GAP analysis aligns two sequencesover their entire length. The polypeptide or class of polypeptides mayhave the same enzymatic activity as, or a different activity than, orlack the activity of, the reference polypeptide. Preferably, thepolypeptide has an enzymatic activity of at least 10% of the activity ofthe reference polypeptide.

As used herein a “biologically active fragment” is a portion of apolypeptide of the invention which maintains a defined activity of afull-length reference polypeptide for example, MGAT activity.Biologically active fragments as used herein exclude the full-lengthpolypeptide. Biologically active fragments can be any size portion aslong as they maintain the defined activity. Preferably, the biologicallyactive fragment maintains at least 10% of the activity of the fulllength polypeptide.

With regard to a defined polypeptide or enzyme, it will be appreciatedthat % identity figures higher than those provided herein will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polypeptide/enzyme comprisesan amino acid sequence which is at least 60%, more preferably at least65%, more preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, more preferably at least 93%, more preferably at least 94%, morepreferably at least 95%, more preferably at least 96%, more preferablyat least 97%, more preferably at least 98%, more preferably at least99%, more preferably at least 99.1%, more preferably at least 99.2%,more preferably at least 99.3%, more preferably at least 99.4%, morepreferably at least 99.5%, more preferably at least 99.6%, morepreferably at least 99.7%, more preferably at least 99.8%, and even morepreferably at least 99.9% identical to the relevant nominated SEQ ID NO.

Amino acid sequence mutants of the polypeptides defined herein can beprepared by introducing appropriate nucleotide changes into a nucleicacid defined herein, or by in vitro synthesis of the desiredpolypeptide. Such mutants include for example, deletions, insertions, orsubstitutions of residues within the amino acid sequence. A combinationof deletions, insertions and substitutions can be made to arrive at thefinal construct, provided that the final polypeptide product possessesthe desired characteristics.

Mutant (altered) polypeptides can be prepared using any technique knownin the art, for example, using directed evolution or rathional designstrategies (see below). Products derived from mutated/altered DNA canreadily be screened using techniques described herein to determine ifthey possess transcription factor, fatty acid acyltransferase or OBCactivities.

In designing amino acid sequence mutants, the location of the mutationsite and the nature of the mutation will depend on characteristic(s) tobe modified. The sites for mutation can be modified individually or inseries for example, by (1) substituting first with conservative aminoacid choices and then with more radical selections depending upon theresults achieved, (2) deleting the target residue, or (3) insertingother residues adjacent to the located site.

Amino acid sequence deletions generally range from about 1 to 15residues, more preferably about 1 to 10 residues and typically about 1to 5 contiguous residues.

Substitution mutants have at least one amino acid residue in thepolypeptide removed and a different residue inserted in its place. Thesites of greatest interest for substitutional mutagenesis to inactivateenzymes include sites identified as the active site(s). Other sites ofinterest are those in which particular residues obtained from variousstrains or species are identical. These positions may be important forbiological activity. These sites, especially those falling within asequence of at least three other identically conserved sites, arepreferably substituted in a relatively conservative manner. Suchconservative substitutions are shown in Table 1 under the heading of“exemplary substitutions”.

TABLE 1 Exemplary substitutions. Original Exemplary ResidueSubstitutions Ala (A) val; leu; ile; gly Arg (R) lys Asn (N) gln; hisAsp (D) glu Cys (C) ser Gln (Q) asn; his Glu (E) asp Gly (G) pro, alaHis (H) asn; gln Ile (I) leu; val; ala Leu (L) ile; val; met; ala; pheLys (K) arg Met (M) leu; phe Phe (F) leu; val; ala Pro (P) gly Ser (S)thr Thr (T) ser Trp (W) tyr Tyr (Y) trp; phe Val (V) ile; leu; met; phe,ala

In a preferred embodiment a mutant/variant polypeptide has only, or notmore than, one or two or three or four conservative amino acid changeswhen compared to a naturally occurring polypeptide. Details ofconservative amino acid changes are provided in Table 1. As the skilledperson would be aware, such minor changes can reasonably be predictednot to alter the activity of the polypeptide when expressed in arecombinant cell. Mutants with desired activity may be engineered usingstandard procedures in the art such as by performing random mutagenesis,targeted mutagenesis, or saturation mutagenesis on known genes ofinterest, or by subjecting different genes to DNA shuffling.

EXAMPLES Example 1. General Materials and Methods

Expression of Genes in Plant Cells in a Transient Expression System

Genes were expressed in plant cells using a transient expression systemessentially as described by Voinnet et al. (2003) and Wood et al.(2009). Binary vectors containing the coding region to be expressed by astrong constitutive e35S promoter containing a duplicated enhancerregion were introduced into Agrobacterium tumefaciens strain AGL1. Achimeric binary vector, 35S:p19, for expression of the p19 viralsilencing suppressor was separately introduced into AGL1, as describedin WO2010/057246. A chimeric binary vector, 35S:V2, for expression ofthe V2 viral silencing suppressor was separately introduced into AGL1.The recombinant cells were grown to stationary phase at 28° C. in LBbroth supplemented with 50 mg/L kanamycin and 50 mg/L rifampicin. Thebacteria were then pelleted by centrifugation at 5000 g for 5 min atroom temperature before being resuspended to OD600=1.0 in aninfiltration buffer containing 10 mM MES pH 5.7, 10 mM MgCl₂ and 100 uMacetosyringone. The cells were then incubated at 28° C. with shaking for3 hours after which the OD600 was measured and a volume of each culture,including the viral suppressor construct 35S:p19 or 35S:V2, required toreach a final concentration of OD600=0.125 added to a fresh tube. Thefinal volume was made up with the above buffer. Leaves were theninfiltrated with the culture mixture and the plants were typically grownfor a further three to five days after infiltration before leaf discswere recovered for either purified cell lysate preparation or totallipid isolation.

Brassica napus Transformation

Brassica napus seeds were sterilized using chlorine gas as described byKereszt et al. (2007) and germinated on tissue culture medium.Cotyledonary petioles with 2-4 mm stalk were isolated as described byBelide et al. (2013) and used as explants. A. tumefaciens AGL1 (Lazo etal., 1991) cultures containing the binary vector were prepared andcotyledonary petioles inoculated with the cultures as described byBelide et al. (2013). Infected cotyledonary petioles were cultured on MSmedium supplemented with 1 mg/L TDZ+0.1 mg/L NAA+3 mg/L AgNO₃+250 mg/Lcefotaxime, 50 mg/L timentin and 25 mg/L kanamycin and cultured for 4weeks at 24° C. with 16 hr/8 hr light-dark photoperiod with a biweeklysubculture on to the same medium. Explants with green callus weretransferred to shoot initiation medium (MS+1 mg/L kinetin+3 mg/LAgNO₃+250 mg/L cefotaxime+50 mg/L timentin+25 mg/L kanamycin) andcultured for another 2-3 weeks. Small shoots (˜1 cm) were isolated fromthe resistant callus and transferred to shoot elongation medium (MSmedium with 0.1 mg/L gibberelic acid+3 mg/L AgNO₃+250 mg/L cefotaxime+25mg/L kanamycin) and cultured for another two weeks. Healthy shoots withone or two leaves were selected and transferred to rooting media (½ MSwith 1 mg/L NAA+20 mg/L ADS+3 mg/L AgNO₃+250 mg/L cefotaxime) andcultured for 2-3 weeks. DNA was isolated from small leaves of resistantshoots using the plant DNA isolation kit (Bioline, Alexandria, NSW,Australia) as described by the manufacturer's protocol. The presence ofT-DNA sequences was tested by PCR amplification on genomic DNA.Positive, transgenic shoots with roots were transferred to potscontaining seedling raising mix and grown in a glasshouse at 24° C.daytime/16° C. night-time (standard conditions).

Purified Leaf Lysate—Enzyme Assays

Nicotiana benthamiana leaf tissues previously infiltrated as describedabove were ground in a solution containing 0.1 M potassium phosphatebuffer (pH 7.2) and 0.33 M sucrose using a glass homogenizer. Leafhomogenate was centrifuged at 20,000 g for 45 minutes at 4° C. afterwhich each supernatant was collected. Protein content in eachsupernatant was measured according to Bradford (1976) using a Wallac1420multi-label counter and a Bio-Rad Protein Assay dye reagent (Bio-RadLaboratories, Hercules, Calif. USA). Acyltransferase assays used 100 μgprotein according to Cao et al. (2007) with some modifications. Thereaction medium contained 100 mM Tris-HCl (pH 7.0), 5 mM MgCl₂, 1 mg/mLBSA (fatty acid-free), 200 mM sucrose, 40 mM cold oleoyl-CoA, 16.4 μMsn-2 monooleoylglycerol[¹⁴C](55 mCi/mmol, American Radiochemicals, SaintLouis, Mo. USA) or 6.0 μM [¹⁴C]glycerol-3-phosphate (G-3-P) disodiumsalt (150 mCi/mmol, American Radiochemicals). The assays were carriedout for 7.5, 15, or 30 minutes.

Lipid Analysis

Analysis of Oil Content in Arabidposis Seeds

When seed oil content or total fatty acid composition was to bedetermined in small seeds such as Arabidopsis seeds, fatty acids in theseeds were directly methylated without crushing of seeds. Seeds weredried in a desiccator for 24 hours and approximately 4 mg of seed wastransferred to a 2 ml glass vial containing a Teflon-lined screw cap.0.05 mg triheptadecanoin (TAG with three C17:0 fatty acids) dissolved in0.1 ml toluene was added to the vial as internal standard. Seed fattyacids were methylated by adding 0.7 ml of IN methanolic HCl (Supelco) tothe vial containing seed material. Crushing of the seeds was notnecessary for complete methylation with small seeds such as Arabidopsisseeds. The mixture was vortexed briefly and incubated at 80° C. for 2hours. After cooling the mixtures to room temperature, 0.3 ml of 0.9%NaCl (w/v) and 0.1 ml hexane was added to the vial and mixed well for 10minutes in a Heidolph Vibramax 110. The FAME were collected into a 0.3ml glass insert and analysed by GC with a flame ionization detector(FID) as described below.

The peak area of individual FAME were first corrected on the basis ofthe peak area responses of a known amount of the same FAMEs present in acommercial standard GLC-411 (NU-CHEK PREP, INC., USA). GLC-411 containsequal amounts of 31 fatty acids (% by weight), ranging from C8:0 toC22:6. In case of fatty acids which were not present in the standard,the peak area responses of the most similar FAME was taken. For example,the peak area response of FAMEs of 16:1d9 was used for 16:1d7 and theFAME response of C22:6 was used for C22:5. The corrected areas were usedto calculate the mass of each FAME in the sample by comparison to theinternal standard mass. Oil is stored mainly in the form of TAG and itsweight was calculated based on FAME weight. Total moles of glycerol wasdetermined by calculating moles of each FAME and dividing total moles ofFAMEs by three. TAG content was calculated as the sum of glycerol andfatty acyl moieties using a relation: % oil by weight=100× ((41× totalmol FAME/3)+(total g FAME−(15× total mol FAME)))/g seed, where 41 and 15are molecular weights of glycerol moiety and methyl group, respectively.

Analysis of Fatty Acid Content in Camelina Seeds and Canola Seeds

To determine fatty acid composition in single seeds that were larger,such as canola and Camelina seeds, direct methylation of fatty acids inthe seed was performed as for Arabidopsis seeds except with breaking ofthe seed coats. This method extracted sufficient oil from the seed toallow fatty acid composition analysis. To determine the fatty acidcomposition of total extracted lipid from seeds, seeds were crushed andlipids extracted with CHCl₃/MeOH. Aliquots of the extracted lipid weremethylated and analysed by GC. Pooled seed-total lipid content (seed oilcontent) of canola was determined by two extractions of lipid usingCHCl₃/MeOH from a known weight of desiccated seeds after crushing,followed by methylation of aliquots of the lipids together with the 17:0fatty acids as internal standard. In the case of Camelina, the lipidfrom a known amount of seeds was methylated together with known amountof 17:0 fatty acids as for the Arabidopsis oil analysis and FAME wereanalysed by GC. For TAG quantitation, TAG was fractionated from theextracted lipid using TLC and directly methylated in silica using 17:0TAG as an internal standard. These methods are described more fully asfollows.

After harvest at plant maturity, Camelina or canola seeds weredesiccated by storing the seeds for 24 hours at room temperature in adesiccator containing silica gel as desiccant. Moisture content of theseeds was typically 6-8%. Total lipids were extracted from known weightsof the desiccated seeds by crushing the seeds using a mixture ofchloroform and methanol (2/1 v/v) in an eppendorf tube using a Reichttissue lyser (22 frequency/seconds for 3 minutes) and a metal ball. Onevolume of 0.1M KCl was added and the mixture shaken for 10 minutes. Thelower non-polar phase was collected after centrifuging the mixture for 5minutes at 3000 rpm. The remaining upper (aqueous) phase was washed with2 volumes of chloroform by mixing for 10 minutes. The second non-polarphase was also collected and pooled with the first. The solvent wasevaporated from the lipids in the extract under nitrogen flow and thetotal dried lipid was dissolved in a known volume of chloroform.

To measure the amount of lipid in the extracted material, a known amountof 17:0-TAG was added as internal standard and the lipids from the knownamount of seeds incubated in 1 N methanolic-HCl (Supelco) for 2 hours at80° C. FAME thus made were extracted in hexane and analysed by GC.Individual FAME were quantified on the basis of the amount of 17:0TAG-FAME. Individual FAME weights, after subtraction of weights of theesterified methyl groups from FAME, were converted into moles bydividing by molecular weights of individual FAME. Total moles of allFAME were divided by three to calculate moles of TAG and thereforeglycerol. Then, moles of TAG were converted in to weight of TAG.Finally, the percentage oil content on a seed weight basis wascalculated using seed weights, assuming that all of the extracted lipidwas TAG or equivalent to TAG for the purpose of calculating oil content.This method was based on Li et al. (2006). Seeds other than Camelina orcanola seeds that are of a similar size can also be analysed by thismethod.

Canola and other seed oil content was also measured by nuclear magneticresonance techniques (Rossell and Pritchard, 1991) by a pulsed wave NMS100 Minispec (Bruker Pty Ltd Scientific Instruments, Germany) asdescribed in Example 14. The NMR method simultaneously measured moisturecontent. Seed oil content can also be measured by near infraredreflectance (NIR) spectroscopy such as using a NIRSystems Model 5000monochromator. Moisture content can also be measured on a sample from abatch of seeds by drying the seeds in the sample for 18 hours at about100° C., according to Li et al. (2006).

Analysis of Lipids from Leaf Lysate Assays

Lipids from the lysate assays were extracted usingchloroform:methanol:0.1 M KCl (2:1:1) and recovered. The different lipidclasses in the samples were separated on Silica gel 60 thin layerchromatography (TLC) plates (MERCK, Dermstadt, Germany) impregnated with10% boric acid. The solvent system used to fractionate TAG from thelipid extract was chloroform/acetone (90/10 v/v). Individual lipidclasses were visualized by exposing the plates to iodine vapour andidentified by running parallel authentic standards on the same TLCplate. The plates were exposed to phosphor imaging screens overnight andanalysed by a Fujifilm FLA-5000 phosphorimager before liquidscintillation counting for DPM quantification.

Total Lipid Isolation and Fractionation of Lipids from VegetativeTissues

Fatty acid composition of total lipid in leaf and other vegetativetissue samples was determined by direct methylation of the fatty acidsin freeze-dried samples. For total lipid quantitation, fatty acids in aknown weight of freeze-dried samples, with 17:0 FFA, were directlymethylated. To determine total TAG levels in leaf samples, TAG wasfractionated by TLC from extracted total lipids, and methylated in thepresence of 17:0 TAG internal standard, because of the presence ofsubstantial amounts of polar lipids in leaves. This was done as follows.Tissues including leaf samples were freeze-dried, weighed (dry weight)and total lipids extracted as described by Bligh and Dyer (1959) or byusing chloroform:methanol:0.1 M KCl (CMK; 2:1:1) as a solvent. Totallipids were extracted from N. benthamiana leaf samples, after freezedying, by adding 900 μL of a chloroform/methanol (2/1 v/v) mixture per 1cm diameter leaf sample. 0.8 μg DAGE was added per 0.5 mg dry leafweight as internal standard when TLC-FID analysis was to be performed.Samples were homogenized using an IKA ultra-turrax tissue lyser afterwhich 500 μL 0.1 M KCl was added. Samples were vortexed, centrifuged for5 min and the lower phase was collected. The remaining upper phase wasextracted a second time by adding 600 μL chloroform, vortexing andcentrifuging for 5 min. The lower phase was recovered and pooled intothe previous collection. Lipids were dried under a nitrogen flow andresuspended in 2 μL chloroform per mg leaf dry weight. Total lipids ofN. tabacum leaves or leaf samples were extracted as above with somemodifications. If 4 or 6 leaf discs (each approx 1 cm² surface area)were combined, 1.6 ml of CMK solvent was used, whereas if 3 or less leafdiscs were combined, 1.2 ml CMK was used. Freeze dried leaf tissues werehomogenized in an eppendorf tube containing a metallic ball using aReicht tissue lyser (Qiagen) for 3 minutes at 20 frequency/sec.

Separation of Neutral Lipids Via TLC and Transmethylation

Known volumes of total leaf extracts such as, for example, 30 μL wereloaded on a TLC silica gel 60 plate (1×20 cm) (Merck KGaA, Germany). Theneutral lipids were fractionated into the different types and separatedfrom polar lipids via TLC in an equilibrated development tank containinga hexane/DEE/acetic acid (70/30/1 v/v/v/) solvent system. The TAG bandswere visualised by primuline spraying, marked under UV, scraped from theTLC plate, transferred to 2 mL GC vials and dried with N₂. 750 μL of INmethanolic-HCl (Supelco analytical, USA) was added to each vial togetherwith a known amount of C17:0 TAG as an internal standard, depending onthe amount of TAG in each sample. Typically, 30 μg of the internalstandard was added for low TAG samples whilst up to 200 μg of internalstandard was used in the case of high TAG samples.

Lipid samples for fatty acid composition analysis by GC weretransmethylated by incubating the mixtures at 80° C. for 2 hours in thepresence of the methanolic-HCl. After cooling samples to roomtemperature, the reaction was stopped by adding 350 μl H₂O. Fatty acylmethyl esters (FAME) were extracted from the mixture by adding 350 μlhexane, vortexing and centrifugation at 1700 rpm for 5 min. The upperhexane phase was collected and transferred into GC vials with 300 μlconical inserts. After evaporation, the samples were resuspended in 30μl hexane. One μl was injected into the GC.

The amount of individual and total fatty acids (TFA) present in thelipid fractions was quantified by GC by determining the area under eachpeak and calculated by comparison with the peak area for the knownamount of internal standard. TAG content in leaf was calculated as thesum of glycerol and fatty acyl moieties in the TAG fraction using arelation: % TAG by weigh=100× ((41× total mol FAME/3)+(total g FAME−(15×total mol FAME)))/g leaf dry weight, where 41 and 15 are molecularweights of glycerol moiety and methyl group, respectively.

Capillary Gas-Liquid Chromatography (GC)

FAME were analysed by GC using an Agilent Technologies 7890A GC (PaloAlto, Calif., USA) equipped with an SGE BPX70 (70% cyanopropylpolysilphenylene-siloxane) column (30 m×0.25 mm i.d., 0.25 μm filmthickness), an FID, a split/splitless injector and an AgilentTechnologies 7693 Series auto sampler and injector. Helium was used asthe carrier gas. Samples were injected in split mode (50:1 ratio) at anoven temperature of 150° C. After injection, the oven temperature washeld at 150° C. for 1 min, then raised to 210° C. at 3° C. min⁻¹ andfinally to 240° C. at 50° C. min⁻¹. Peaks were quantified with AgilentTechnologies ChemStation software (Rev B.04.03 (16), Palo Alto, Calif.,USA) based on the response of the known amount of the external standardGLC-411 (Nucheck) and C17:0-Me internal standard.

Quantification of TAG Via Latroscan

One μL of lipid extract was loaded on one Chromarod-SII for TLC-FIDIatroscan™ (Mitsubishi Chemical Medience Corporation—Japan). TheChromarod rack was then transferred into an equilibrated developing tankcontaining 70 mL of a hexane/CHCl₃/2-propanol/formic acid(85/10.716/0.567/0.0567 v/v/v/v) solvent system. After 30 min ofincubation, the Chromarod rack was dried for 3 min at 100° C. andimmediately scanned on an latroscan MK-6s TLC-FID analyser (MitsubishiChemical Medience Corporation—Japan). Peak areas of DAGE internalstandard and TAG were integrated using SIC-48011 integration software(Version:7.0-E SIC System instruments Co., LTD—Japan).

TAG quantification was carried out in two steps. First, DAGE was scannedin all samples to correct the extraction yields after which concentratedTAG samples were selected and diluted. Next, TAG was quantified indiluted samples with a second scan according to the external calibrationusing glyceryl trilinoleate as external standard (Sigma-Aldrich).

Quantification of TAG in Leaf Samples by GC

The peak area of individual FAME were first corrected on the basis ofthe peak area responses of known amounts of the same FAMEs present in acommercial standard GLC-411 (NU-CHEK PREP, Inc., USA). The correctedareas were used to calculate the mass of each FAME in the sample bycomparison to the internal standard. Since oil is stored primarily inthe form of TAG, the amount of oil was calculated based on the amount ofFAME in each sample. Total moles of glycerol were determined bycalculating the number of moles of FAMEs and dividing total moles ofFAMEs by three. The amount of TAG was calculated as the sum of glyceroland fatty acyl moieties using the formula: % oil by weight=100× ((41×total mol FAME/3)+(total g FAME-(15× total mol FAME))/g leaf dry weight,where 41 and 15 were the molecular weights of glycerol moiety and methylgroup, respectively.

Example 2. Increasing Lipid Content in Nicotiana benthamiana VegetativeParts

The genetic construct pJP3502 was used to produce stably transformedplants of Nicotiana benthamiana by the Agrobacterium-mediatedtransformation protocol as described for Nicotiana tabacum inWO2013/096993. Transgenic plants were selected for kanamycin resistanceand grown to maturity in the glasshouse. Leaf samples were harvested atseed set and freeze-dried. Total fatty acid (TFA) content (% of drymass) and composition following Bligh and Dyer (1959) extraction oftotal lipids from the samples, and the triacylglycerol (TAG) fractioncontent and composition, were determined. Data are shown in Table 2 andTable 3. The highest leaf oil sample was from transgenic plant #16 whichhad a TFA content of 33% by weight. This sample contained 22.5% TAG byweight (dry weight).

A strong correlation between alterations in the fatty acid compositionand the TFA or TAG contents was observed. Oleic acid (C18:1n-9)increased with increasing TFA and TAG contents, so that it was thedominant fatty acid in leaves with high TAG content, for examplecomprising 66.8% of the TFA and 66.9% of the TAG fatty acids in theleaves with the highest TAG content. Similar correlations were observedfor other fatty acids, for example ALA levels were reduced to 4.9% ofTFA and 3.9% of TAG in the leaves with the highest TAG content. A strongcorrelation between C16:3 levels and both TFA and TAG contents was alsoobserved with C16:3 decreasing substantially in high TFA and TAGsamples.

Two of the high oil plants, #14 and #16, were also analysed during theleaf senescence phase when the leaves had begun yellowing (Table 4 andTable 5). Whilst there was little change in the total fatty acid contentof the highest sample (32.9% vs 33%) the amount of TAG had increased to32.6%. In these samples TAG comprised almost all of the leaf lipids.

TABLE 2 Total fatty acid (TFA) composition and amount (% dry weight) inleaves of Nicotiana benthamiana plants stably transformed with the T-DNAfrom construct pJP3502. The samples also contained 0.1-0.3% C14:0 and0.0-0.7% C20:1 Sample C16:0 C16:1 C16:3 C18:0 C18:1 C18:1d11 C18:2C18:3n3 C20:0 C22:0 C24:0 % TFA Controls 1 24.9 0.9 6.9 4.2 11.7 0.0 2.646.4 0.8 0.6 0.8 0.6 2 23.9 0.9 12.0 4.8 13.4 0.0 2.8 39.1 0.9 0.6 1.30.6 3 24.1 0.9 10.4 4.7 10.5 0.0 2.8 43.7 0.9 0.6 1.2 0.5 4 24.0 0.910.0 4.7 13.7 0.0 2.9 40.8 0.9 0.6 1.2 0.7 Transgenics 7 18.6 0.2 2.34.2 35.8 0.3 4.7 28.0 2.3 1.5 1.3 2.4 8 16.3 0.6 0.7 4.9 62.9 0.7 4.16.6 1.4 0.7 0.6 21.8 11 25.9 1.2 2.8 3.2 47.3 0.8 3.1 13.3 1.3 0.7 0.114.7 12 24.7 1.1 1.6 3.2 46.0 1.1 3.1 16.8 1.1 0.6 0.2 3.8 13 20.3 0.621.1 4.7 15.0 0.0 2.9 33.4 1.2 0.0 0.6 1.2 14 15.6 0.5 0.6 5.1 64.4 0.62.7 6.5 1.8 0.9 0.7 21.3 15 17.7 0.4 0.2 5.6 60.7 0.4 2.7 7.5 2.2 1.20.9 21.0 16 15.3 0.6 0.6 5.8 66.8 0.6 1.7 4.9 1.7 0.8 0.7 33.0 17 25.50.0 7.4 4.2 8.6 0.0 3.7 48.8 0.7 0.0 0.9 1.4 18 27.0 0.6 5.7 2.5 6.7 2.05.1 48.7 0.5 0.4 0.6 2.6 19 21.1 0.8 2.1 5.2 35.9 1.0 10.0 20.8 1.5 0.90.2 4.3 20 15.4 1.8 4.6 3.4 10.5 0.0 5.6 56.4 0.8 0.5 0.8 2.3 21 16.30.7 6.6 3.6 10.2 0.0 10.1 49.7 1.4 0.8 0.7 1.6

TABLE 3 Fatty acid composition and amount (% dry weight) of the TAG inleaves of Nicotiana benthamiana plants stably transformed with the T-DNAfrom the construct pJP3502. The samples also contained 0.1-0.3% C14:0and 0.0-0.7% C20:1 Sample C16:0 C16:1 C16:3 C18:0 C18:1 C18:1d11 C18:2C18:3n3 C20:0 C22:0 C24:0 % TAG Control 1 57.7 0.0 0.0 6.6 7.4 0.0 0.028.3 0.0 0.0 0.0 0.1 2 61.7 0.0 1.8 8.1 7.5 0.0 1.9 19.0 0.0 0.0 0.0 0.13 69.9 0.0 0.0 8.7 6.0 0.0 0.0 15.5 0.0 0.0 0.0 0.1 4 59.2 0.0 1.1 7.68.8 0.0 2.1 18.2 1.3 0.0 1.7 0.2 Transgenics 7 26.7 0.2 1.0 6.1 38.1 0.43.8 16.8 3.6 2.1 0.2 2.5 8 17.3 0.7 0.2 5.3 64.4 0.8 2.9 5.1 1.5 0.7 0.615.4 11 28.9 1.5 0.2 3.4 49.9 1.0 2.6 9.3 1.3 0.7 0.8 9.7 12 27.0 1.40.3 3.4 51.4 1.4 2.6 9.6 1.2 0.6 0.7 5.2 13 39.7 1.3 4.0 5.5 17.2 0.02.4 27.9 1.0 0.0 0.6 0.6 14 16.2 0.6 0.2 5.3 65.1 0.6 2.8 5.2 1.8 0.90.7 19.6 15 18.4 0.4 0.1 5.9 61.0 0.4 2.9 5.8 2.3 1.3 1.0 14.9 16 15.90.7 0.2 5.9 66.9 0.7 2.1 3.9 1.7 0.8 0.7 22.5 17 29.8 0.0 0.0 4.2 13.50.0 5.1 47.4 0.0 0.0 0.0 0.4 18 40.2 5.9 0.0 3.2 10.8 2.0 5.6 32.4 0.00.0 0.0 0.6 19 24.6 1.0 0.7 6.8 43.6 1.1 9.4 8.7 1.9 1.0 0.8 3.7 20 23.10.0 1.1 6.0 18.6 0.0 7.6 41.5 2.1 0.0 0.0 0.6 21 28.1 0.0 2.5 6.2 19.90.0 11.5 27.7 2.7 1.3 0.0 0.7

TABLE 4 Yellow leaf stage total fatty acid (TFA) composition and amount(% dry weight) in leaves from Nicotiana benthamiana plants stablytransformed with the T-DNA from the construct pJP3502. The samples alsocontained 0.1-0.3% C14:0 and 0.0-0.7% C20:1 Sample C16:0 C16:1 C16:3C18:0 C18:1 C18:1d11 C18:2 C18:3n3 C20:0 22:0 C24:0 % TFA Control 1 24.90.9 6.9 4.2 11.7 0.0 2.6 46.4 0.8 0.6 0.8 0.6 Transgenic 14 17.2 0.5 0.76.0 64.7 0.6 0.1 6.8 2.1 0.0 0.6 29.0 16 17.4 0.6 0.8 7.0 64.7 0.7 0.15.5 1.9 0.0 0.6 32.9

TABLE 5 Yellow leaf stage fatty acid composition and amount (% dryweight) of TAG in leaves of Nicotiana benthamiana plants stablytransformed with the T-DNA from the construct pJP3502. The samples alsocontained 0.1-0.3% C14:0 and 0.0-0.7% C20:1 Sample C16:0 C16:1 C16:3C18:0 C18:1 C18:1d11 C18:2 C18:3n3 C20:0 22:0 C24:0 % TAG Control 1 38.10.0 0.0 7.0 10.6 0.0 2.9 40.3 1.1 0.0 0.0 0.1 Transgenic 14 16.9 0.5 0.25.5 62.8 0.6 3.0 6.5 1.9 0.9 0.6 27.7 16 17.3 0.7 0.2 6.4 63.3 0.6 2.25.5 1.8 0.8 0.6 32.6

Example 3. Increasing Lipid Content in Vegetative Nicotiana tabacumPlant Parts

The construct pJP3502 had previously been used to transform Nicotianatabacum (WO2013/096993). Seed obtained from a homozygous T1 planttransformed with the T-DNA from pJP3502 and having high TFA and TAGcontent was harvested and sown out to establish a new generation of T2progeny plants, uniformly homozygous for the transgenes. Pots werearranged in the glasshouse such that mature plant leaves eitheroverlapped in a typical canopy formation (‘canopy’) as would occur whengrown in the field, or were maximally exposed to direct sunlight(‘non-canopy’). Leaf samples were taken from each plant when fullygrown, at seed-setting stage, and freeze-dried. Fatty acid content wasdetermined for the TAG fraction (Table 6) following Bligh and Dyer(1959) extraction of total lipids from the samples. TAG levels in matureleaf tissue from non-canopy plants were typically higher than for canopyplants, with the highest observed leaf TAG content of 20.6% of leaf dryweight.

TABLE 6 TAG content (% dry weight) in mature leaf tissue of T2transgenic progeny plants (Line 49) transformed with T-DNA of pJP3502,compared to wild-type (wt). Growing TAG Growing TAG Plant conditioncontent Plant condition content wt 1 Canopy 0.0 wt 4 Non-canopy 0.0 wt 2Canopy 0.1 wt 5 Non-canopy 0.0 wt 3 Canopy 0.1 49.6 Non-canopy 5.1 49.1Canopy 6.4 49.7 Non-canopy 5.6 49.2 Canopy 3.6 49.8 Non-canopy 14.7 49.3Canopy 3.7 49.9 Non-canopy 6.3 49.4 Canopy 1.9 49.10 Non-canopy 6.7 49.5Canopy 2.2 49.11 Non-canopy 19.5 49.12 Non-canopy 16.4 49.13 Non-canopy20.6 49.14 Non-canopy 15.7 49.15 Non-canopy 15.1 49.16 Non-canopy 6.349.17 Non-canopy 18.6

Example 4. Increasing Oil Content in Vegetative Parts ofMonocotyledonous Plants

Chimeric DNA constructs were designed to increase oil content inmonocotyledonous plants, for example the C4 plant S. bicolor (sorghum),by expressing a combination of genes encoding WRI1, Z. mays LEC1(Accession number AAK95562; SEQ ID NO:155), DGAT and Oleosin in thetransgenic plants. Several pairs of constructs for biolisticco-transformation were designed and produced by restrictionenzyme-ligation cloning, as follows.

The genetic construct pOIL136 was a binary vector containing threemonocot expression cassettes, namely a selectable marker gene encodingphosphinothricin acetyltransferase (PAT) for plant selection, a secondcassette for expressing DGAT and a third for expressing Oleosin. pJP136was first produced by amplifying an actin gene promoter from Oryzasativa (McElroy et al., 1990) and inserting it as a blunt-ClaI fragmentinto pORE04 (Coutu et al., 2007) to produce pOIL094. pOIL095 was thenproduced by inserting a version of the Sesamum indicum Oleosin genewhich had been codon optimised for monocot expression into pOIL094 atthe KpnI site. pOIL093 was produced by cloning a monocot codon optimisedversion of the Umbelopsis ramanniana DGAT2a gene (Lardizabal et al.,2008) as a SmaI-KpnI fragment into a vector already containing a Zeamays Ubiquitin gene promoter. pOIL134 was then produced by cloning theNotI DGAT2a expression cassette from pOIL093 into pOIL095 at the Nodsites. pOIL141 was produced by inserting the selectable marker genecoding for PAT as a BamHI-SacI fragment into a vector containing the Z.mays Ubiquitin promoter. Finally, pOIL136 was produced by cloning the Z.mays Ubiquitin::PAT expression cassette as a blunt-AscI fragment intothe ZraI-AscI of pOIL096. The genetic construct pOIL136 thereforecontained the following expression cassettes: promoter O. sativaActin::S. indicum Oleosin, promoter Z. mays Ubiquitin::U. ramannianaDGAT2a and promoter Z. mays Ubiquitin::PAT.

A similar vector pOIL197, containing NPTII instead of PAT wasconstructed by subcloning of the Z. mays Ubiquitin::NPTII cassette frompUKN as a HindIII-SmaI fragment into the AscI (blunted) and HindIIIsites of pJP3343. The resulting vector, pOIL196, was then digested withHindIII (blunted) and AgeI. The resulting 3358 bp fragment was clonedinto the ZraI-AgeI sites of pOIL134, yielding pOIL197.

A set of constructs containing genes encoding the Z. mays WRI1 (ZmWRI)or the LEC1 (ZmLEC1) transcription factors under the control ofdifferent promoters were designed and produced for biolisticco-transformation in combination with pOIL136 to test the effect ofpromoter strength and cell specificity on the function of WRI1 or LEC1,or both if combined, when expressed in vegetative tissues of a C4 plantsuch as sorghum. This separate set of constructs did not contain aselectable marker gene, except for pOIL333 which contained NPTII asselectable marker. The different promoters tested were as follows. TheZ. mays Ubiquitin gene promoter (pZmUbi) was a strong constitutivemonocot promoter while the enhanced CaMV 35S promoter (e35S) having aduplicated enhancer region was reported to result in lower transgeneexpression levels (reviewed in Girijashankar and Swathisree, 2009).Whilst the Z. mays phosphoenolpyruvate carboxylase (pZmPEPC) genepromoter was active in leaf mesophyl cells (Matsuoka and Minami, 1989),the site of photosynthesis in C4 plant species, the Z. mays Rubiscosmall subunit (pZmSSU) gene promoter was specific for the bundle sheathcell layer (Nomura et al., 2000; Lebrun et al., 1987), the cells wherecarbon fixation takes place in C4 plants.

The expression of the Z. mays gene encoding the SEE1 cysteine protease(Accession number AJ494982) was identified as similar to that of the A.thaliana SAG12 senescence-specific promoter during plant development.Therefore a 1970 bp promoter from the SEE1 gene (SEQ ID NO:216) was alsoselected to drive expression of the genes encoding the Z. mays WRI1 andLEC1 transcription factors. Further, the promoter from the gene encodingAeluropus lilttoralis zinc finger protein AlSAP (Ben Saad et al., 2011;Accession number DQ885219; SEQ ID NO:217) and the promoter from thesucrose-responsive ArRolC gene from A. rhizogenes (Yokoyama et al.,1994; Accession number DQ160187; SEQ ID NO:218) were also selected forexpression of ZmWRI1 expression in stem tissue. Therefore, each of thesepromoters was individually joined upstream of the ZmWRI1 or ZmLEC1coding regions, as follows.

An intermediate vector, pOIL100, was first produced by cloning the Z.mays WRI1 coding sequence and a transcription terminator/polyadenylationregion, flanked by AscI-NcoI sites, into the same sites in the binaryvector pJP3343. The different versions of the constructs for WRI1expression were based on this vector and were produced by cloning thevarious promoters into pOIL100. pOIL101 was produced by cloning aXhoI-SalI fragment containing the e35S promoter with duplicated enhancerregion into the XhoI site of pOIL100. pOIL102 was produced by cloning aHindIII-AvrII fragment containing the Z. mays Ubiquitin gene promoterinto the HindIII-XbaI sites of pOIL100. pOIL103 was produced by cloninga HindIII-NcoI fragment containing a Z. mays PEPC gene promoter into theHindIII-NcoI sites of pOIL100. pOIL104 was produced by cloning aHindIII-AvrII fragment containing a Z. mays SSU gene promoter into theHindIII-AvrII sites of pOIL100.

A synthetic fragment containing the Z. mays SEE1 promoter region flankedby HindIII-XhoI unique sites is synthesized. This fragment is clonedupstream of the Z. mays WRI1 protein coding region using theHindIII-XhoI sites in pOIL100. The resulting vector is designatedpOIL329. A synthetic fragment containing the A. littoralis AlSAPpromoter region flanked by XhoI-XbaI unique sites is synthesized. Thisfragment is cloned upstream of the Z. mays WRI1 coding region using theXbaI-XhoI sites in pOIL100. The resulting vector is designated pOIL330.A synthetic fragment containing the A. rhizogenes ArRoIC promoter regionflanked by PspOMI-XhoI unique sites is synthesized. This fragment iscloned upstream of the Z. mays WRI1 coding region using the PspOMI-XhoIsites in pOIL100. The resulting vector is designated pOIL335. Finally, abinary vector (pOIL333) containing the Z. mays SEE1::ZmLEC1 expressioncassette is obtained in three steps. First, a 35S::GUS expression vectoris constructed by amplifying the GUS coding region with flanking primerscontaining AvrII and KpnI sites. The resulting fragment is subsequentlycloned into the SpeI-KpnI sites of pJP3343. The resulting vector isdesignated pTV111. Next, the 35S promoter region of pTV111 is replacedby the Z. mays SEE1 promoter. To this end, the Z. mays SEE1 sequence isamplified using flanking primers containing HindIII and XhoI uniquesites. The resulting fragment is cut with the respective restrictionenzymes and subcloned into the SalI-HindIII sites of pTV111. Theresulting vector is designated pOIL332. Next the ZmLEC1 coding sequenceis amplified using flanking primers containing NotI and EcoRV sites.This resulting fragment is subcloned into the respective sites ofpOIL332, yielding pOIL333.

DNA is prepared for biolistic transformation by excising the vectorbackbones from pOIL101, pOIL102, pOIL103, pOIL04, pOIL197, pOIL329,pOIL330, pOIL333 and pOIL335 by restriction digestion followed by gelisolation. pOIL197 DNA is then mixed with either pOIL101, pOIL102,pOIL103, pOIL104, pOIL329, pOIL330, pOIL333 or pOIL335 DNA andtransformed by biolistic-mediated transformation into S. bicolorexplants. Alternatively, constructs for expression of the samecombinations of genes are transformed separately or co-transformed byAgrobacterium-mediated transformation (Gurel et al., 2009; Wu et al.,2014).

Transgenic plants are regenerated and selected by antibiotic resistance.Where the two constructs co-transform in the same event, increased oilcontent is observed in the non-seed tissues of the transgenic plants.

The chimeric DNA constructs for Agrobacterium-mediated transformationare used to transform Zea mays (corn) as described by Gould et al.,(1991). Briefly, shoot apex explants are co-cultivated with transgenicAgrobacterium for two days before being transferred onto a MS salt mediacontaining kanamycin and carbenicillin. After several rounds ofsub-culture, transformed shoots and roots spontaneously form and aretransplanted to soil. The constructs are similarly used to transformHordeum vulgare (barley) and Avena sativa (oats) using transformationmethods known for these species. Briefly, for barley, the Agrobacteriumcultures are used to transform cells in immature embryos of barley (cv.Golden Promise) according to published methods (Tingay et al., 1997;Bartlett et al., 2008) with some modifications in that embryos between1.5 and 2.5 mm in length are isolated from immature caryopses and theembryonic axes removed. The resulting explants are co-cultivated for 2-3days with the transgenic Agrobacterium and then cultured in the dark for4-6 weeks on media containing timentin and hygromycin to generateembryogenic callus before being moved to transition media in low lightconditions for two weeks. Calli are then transferred to regenerationmedia to allow for the regeneration of shoots and roots before transferof the regenerated plantlets to soil. Transformed plants are obtainedand grown to maturity in the glasshouse.

Example 5. Increasing Oil Content in Dicotyledonous Plants

Oil content in the dicotyledonous plant species Trifolium repens(clover), a legume commonly used as a pasture species, was increased byexpressing the combination of WRI1, DGAT and Oleosin genes in vegetativeparts. The construct pJP3502 was used to transform T. repens byAgrobacterium-mediated transformation (Larkin et al., 1996). Briefly,the genetic construct pJP3502 was introduced into A. tumefaciens via astandard electroporation procedure. The binary vector also contained a35S:NptII selectable marker gene within the T-DNA. The transformedAgrobacterium cells were grown on solid LB media supplemented withkanamycin (50 mg/L) and rifampicin (25 mg/L) and incubated at 28° C. fortwo days. A single colony was used to initiate a fresh culture.Following 48 hours vigorous culture, the Agrobacterium cells was used totreat T. repens (cv. Haifa) cotyledons that had been dissected fromimbibed seed as described by Larkin et al. (1996). Followingco-cultivation for three days the explants were exposed to 25 mg/Lkanamycin to select transformed shoots and then transferred to rootingmedium to form roots, before transfer to soil.

Six transformed plants containing the T-DNA from pJP3502 were obtainedand transferred to soil in the glasshouse. Increased oil content wasobserved in the non-seed tissue of some of the plants, with one plantshowing greater than 4-fold increase in TAG levels in the leaves. Suchplants are useful as animal feed, for example by growing the plants inpastures, providing feed with an increased energy content per unitweight (energy density) and resulting in increased growth rates in theanimals.

The construct pJP3502 is also used to transform other leguminous plantssuch as alfalfa (Medicago sativa) and barrel medic (Medicago truncatula)by the method of Wright et al. (2006) to obtain transgenic plants whichhave increased TAG content in vegetative parts. Three putativetransgenic M. truncatula plants were obtained. The transgenic plants areuseful as pasture species or as hay or silage as a source of feed foranimals such as, for example, cattle, sheep and horses, providing anincreased energy density in the feed.

For increasing the oil content in legume seeds, a DNA fragment wassynthesised containing a combination of two chimeric genes, namely (a) afirst chimeric gene encoding A. thaliana WRI1 expressed from thePhaseolus vulgaris beta-type phaseolin storage protein promoter and 5′UTR plus (b) a second chimeric gene encoding A. thaliana DGAT1 expressedfrom a Pisum sativum vicilin promoter and 5′ UTR. The DNA fragment wasinserted into a binary vector pORE04 containing a chimeric gene encodingoleosin to generate a T-DNA construct comprising the three chimericgenes and a selectable marker gene (FIG. 2) which was used to transformLupinus angustifolius, another leguminous plant, by the method asdescribed by Pigeaire et al. (1997). Briefly, shoot apex explants of L.angustifolius are co-cultivated with transgenic Agrobacterium beforebeing thoroughly wetted with kanamycin solution (20 mg/ml) andtransferred onto a kanamycin-free regeneration medium. The multipleaxillary shoots developing from the shoot apices are excised onto amedium containing 50 mg/L kanamycin and the surviving shoots transferredonto fresh medium containing 50 mg/L kanamycin. Healthy shoots are thentransferred to soil. The genes on the T-DNA are expressed in cells ofthe transformed plants, increasing the oil content in the vegetativetissues and the seeds. A seed specific promoter driving the WRI1 gene isalso used to increase the oil content in transgenic Lupinus seeds.

The construct was also used to transform Glycine max as described byZhang et al. (1999) to obtain transgenic soybean plants which haveincreased TAG content in seeds. Transgenic plants were obtained asdemonstrated by PCR on DNA obtained from samples of the plants. Theplants were grown to maturity and seed was harvested from the. The oilcontent of the seed is expected to be increased as determined bynon-destructive NMR.

A second genetic construct for increasing seed oil content in lupin andsoybean was constructed by synthesising a DNA insert comprising threegene expression cassettes, namely a first having a Glycine maxβ-conglycinin promoter expressing Umbelopsis ramanniana DGAT2A, a secondhaving a Glycine max KTi3 promoter expressing A. thaliana WRI1 and athird having the Glycine max β-conglycinin promoter expressing Musmusculus MGAT2. The SbfI-PstI fragment of this insert was cloned intothe binary vector pORE04 at the PstI site to yield pJP3569. A versionwithout the MGAT2 gene was made by cloning the smaller SbfI-SwaIfragment into pORE04 at the EcoRV-PstI sites to yield pJP3570. Versionscontaining only the WRI1 gene and only the DGAT2A gene were similarlyproduced. These binary vectors were used to transform Glycine max togenerate transgenic seed. The oil content in the seed from the primarytransformants is analysed by non-destructive NMR before being sown outto produce T2 seed.

A version of pJP3569 suitable for lupin transformation is made by PCRamplifying the WRI1 and DGAT2A expression cassettes in a single ampliconadapted with NotI restriction sites. The NotI fragment is cloned intopJP3416 at the PspOMI site to yield pJP3678, a binary vector containingthe PAT selectable marker gene.

Example 6. Experiments to Increase Oil Content in Vegetative Parts ofCanola

Two binary expression vectors were used to transform B. napus (cv.Oscar) in order to investigate the effect on TAG accumulation in seedand/or vegetative tissues. Firstly, the plasmid pJP3414 was constructedby inserting a codon optimised A. thaliana WRI1 protein coding sequenceinto binary vector 35S-pORE04 which contained an empty 35S expressioncassette. The T-DNA of pJP3414 therefore containing a codon optimizedversion of the A. thaliana WRI1 transcription factor under the controlof the constitutive 35S promoter. Leaf tissue from 11 independentlytransformed B. napus T0 seedlings, transformed with pJP3414 as describedin Example 1, each contained elevated TAG levels compared to the emptyvector (pORE04) transformed plants. However, in no case did the level ofTAG exceed 1%. Maximum levels were detected in line 31 which containedup to 0.58% TAG on a dry weight basis. The oil content in T1 transgenicseed was not significantly elevated compared to wild type (Oscar) andempty vector control seeds. T1 seeds of three lines exhibiting thehighest TAG levels in leaf tissue were germinated on MS media containing3% sucrose. No difference in germination was observed after 5 and 8 dayswhen compared to the untransformed control (Oscar).

In an attempt to further increase TAG levels in B. napus vegetativetissues, the second vector, pJP3502 (Vanhercke et al., 2014), was usedto transform B. napus (cv. Oscar). TAG levels were quantitated intransgenic leaves sampled before flowering. However, the TAG content wasnot further increased and the fatty acid composition did not differ fromuntransformed control plants at this stage of growth.

The observations for B. napus described in this Example, providing a TAGcontent of less than 1% in leaves, were in stark contrast to thosereported in Examples 2 and 3 for Nicotiana species, providing about20-30% TAG. The inventors considered these observations thoroughly,seeking an explanation for the difference between the species. Severaldifferences were identified between the species. One difference that theinventors conceived as providing the essential difference was thatBrassica napus is a so-called 16:3 species whereas Nicotiana species areso-called 18:3 species. This relates to the relative contribution of theso-called prokaryotic and eukaryotic pathways for plastid lipidsynthesis (FIG. 1), and therefore, the inventors thought, the amount ofDAG that is available for TAG synthesis. This led the inventors toconceive of the model that modification of the ratio of the synthesis offatty acids via the eukaryotic pathway relative to the prokaryoticpathway, for example by decreasing the accumulation of 16:3 relative to18:3, would alter the level of TAG that accumulates in plant cells orphotosynthetic, microbial cells. They expected such modification thattipped the balance in favour of the eukaryotic pathway would beadvantageous for TAG accumulation levels, especially in so-called 16:3plants. In short, to convert a “16:3” cell into more like an “18:3”cell.

The inventors hypothesized that the presence of C18:1-ACP in the plastidwhich inhibits ACCase by feedback inhibition could be stronger in 16:3plants due to the synthesis and retention of fatty acids in the plastidby the prokaryotic pathway. In contrast, they hypothesized that C18:3plants are capable of accumulating higher TAG levels in vegetativetissues due to increased C18:1 export out of the plastids for provisioninto the eukaryotic pathway. As shown in Examples 2 to 4 herein, thiswas observed in species such as N. tabacum and N. benthamiana which havehigher C18:3/C16:3 plastidial lipid ratios relative to species such asB. napus which has low levels of C18:3 in plastidial lipids. This model,the inventors hypothesized, would explain why stable transformation ofthe WRI+DGAT+Oleosin expression genes from vector pJP3502 into both N.tabacum and N. benthamiana resulted in high levels of TAG accumulationand extensive changes in fatty acid composition. In contrast,transformation of the same vector into B. napus resulted in only a minorincrease in TAG accumulation and a small change in fatty acidcomposition. This model was examined as described below.

Example 7. Modification of Plastidial GPAT Expression

Over-Expression of Plastidial GPAT in Plant Cells

A number of experiments were performed to test the hypothesis that thepresence of a highly active 16:3 prokaryotic pathway in a plant (i.e. aso-called 16:3 plant) would provide much lower TAG levels in vegetativetissues upon introduction of the gene combination on pJP3502, relativeto 18:3 plants. These experiments are described in the followingExamples. Initially, the inventors tested whether the high level TAGaccumulation observed in transgenic N. benthamiana could be disrupted byover-expression of a plastidial GPAT, increasing the flux in theprokaryotic pathway.

A coding region for expression of the Arabidopsis thaliana plastidialGPAT, ATS1 (Nishida et al., 1993), was amplified by RT-PCR from A.thaliana total RNA and cloned as an EcoRI-PstI fragment into the binaryexpression vector pJP3343 under the control of the 35S promoter toproduce the constitutive expression vector pOIL098. The effect ofover-expressing a plastidial GPAT in a high oil leaf background isdetermined by infiltration of the chimeric vector pOIL098 into high oilleaf tissue. The high oil leaf tissue is generated either byco-infiltration of WRI1 and DGAT binary expression vectors (Example 1)or by infiltrating pOIL098 into leaves of a Nicotiana plant stablytransformed with the T-DNA from pJP3502 or another high oil vector. Oilcontent is expected to be reduced in the infiltrated leaf spotsco-expressing the ATS1-encoding gene. This is determined by analysingTFA and TAG as proportions of sample dry mass. This is also determinedby observing incorporation of labelled acetate into fatty acids producedby microsomes or leaf lysates made from infiltrated leaf spots.

Oil Accumulation in a Plastidial GPAT Mutant of Arabidopsis thaliana

The ats1 mutant of A. thaliana has a disruptive mutation in the geneencoding plastidial GPAT which reduced plastidial GPAT activity to alevel of only 3.8% of the wild-type (Kunst et al., 1988). Non-seed TAGaccumulation levels, at least in leaves, stems and roots, in bothparental and ats1 mutant A. thaliana is tested and compared. The T-DNAof the pJP3502 construct for over-expression of the combination of genesencoding WRI1, DGAT and Oleosin is introduced by transformation intoplants of both genotypes. The gene combination in the T-DNA of pJP3502increases fatty acid synthesis in both plant backgrounds. However, theaccumulation of TAG in the ats1 mutant is expected to be significantlyhigher on average than in the transgenic plants derived from thewild-type (parental) genotype due to the reduction in plastidial GPATactivity and therefore the reduced flux of fatty acids into theplastidial prokaryotic pathway. The ratio of the fatty acids C16:3 toC18:3 is significantly reduced in leaves of the ats1 mutant, bothtransformed and untransformed.

Silencing the Gene Encoding Plastidial GPAT in Plant Cells

In addition to genetically modifying a plant by introducing a mutationin a gene encoding a plastidial GPAT, the flux of fatty acids throughthe prokaryotic 16:3 pathway can be reduced and thereby increase oilcontent in vegetative parts by silencing the plastidial GPAT. This isdemonstrated by producing a transgenic cassette having a constitutive orleaf-specific promoter expressing an RNA hairpin corresponding to aregion of the gene encoding the plastidial GPAT from the selectedspecies. As an example, an RNAi hairpin expression cassette is producedusing the 581 bp SalI-EcoRV fragment of the A. thaliana plastidial GPATcDNA sequence (NM_179407, SEQ ID NO:177). A region of any gene encodinga plastidial GPAT which has a high degree of sequence identity to thenucleotide sequence of NM_179407 can also be used to construct a genefor expression of a hairpin RNA for silencing an endogenous plastidialGPAT gene. A hpRNAi construct containing a 732 bp fragment (SEQ IDNO:219) of the N. benthamiana plastidial GPAT flanked by SmaI and KasIunique sites was designed for stable transformation into N. tabacum. Thesynthesized N. benthamiana plastidial GPAT fragment was subcloned intothe SmaI-KasI sites of pJP3303, resulting in pOIL113. It is expectedthat reducing plastidial fatty acid retention will result in an increasein TAG accumulation, particularly when combined with a “Push” componentsuch as over-expression of a transcription factor such as WRI1, or by a“Pull” component such as a DGAT or PDAT, and/or reduced SDP1 or TGDactivity.

Inactivation of the gene encoding a plastidial GPAT or indeed any genecan be achieved using CRISPR/Cas9 methods. For example, inactivation ofthe gene encoding A. thaliana plastidial GPAT (Accession No. NM_179407)can be carried out by CRISPR/Cas9/sgRNA-mediated gene disruption andsubsequent mutagenesis by non homologous end joining (NHEJ) DNA repair.Before targeted DNA cleavage, Cas9 stimulates DNA strand separation andallows a sgRNA to hybridize with a specific 20 nt sequence in thetargeted gene. This positions the target DNA into the active site ofCas9 in proper orientation in relation to a PAM (tandem guanosinenucleotides) binding site. This positioning allows separate nucleasedomains of Cas9 to independently cleave each strand of the target DNAsequence at a point 3-nt upstream of the PAM site. The double-strandbreak then undergoes error-prone NHEJ DNA repair during which deletionsor insertions of a few nucleotides occur and result in inactivation ofthe plastidial GPAT gene. SgRNA sequences targeting the A. thaliana GPATgene are identified and selected through the use of the CRISPRP web tool(Xie et al., 2014). The 20 nt target sequence can be any 20 nt sequencewithin the target gene, including within non-coding regions of the genesuch as a promoter or intron, provided that it is a specific sequencewithin the genome. The sequence can be inserted into a binary vectorcontaining the CRISPR/Cas9/sgRNA expression cassette and kanamycin plantselectable marker (Jiang et al., 2013) and transformed into the plantcells by Agrobacterium-mediated transformation. Transgenic T1 plants canbe screened for mutations in the plastidial GPAT gene by PCRamplification and DNA sequencing.

Example 8. Increasing Expression of Thioesterase in Plant Cells

De novo fatty acid synthesis takes place in the plastids of eukaryoticcells where the fatty acids are synthesized while bound to acyl carrierprotein as acyl-ACP conjugates. Following chain elongation to C16:0 andC18:0 acyl groups and then desaturation to C18:1 while linked to ACP,the fatty acids are cleaved from the ACP by thioesterases and enter theeukaryotic pathway by export from the plastids and transport to the ERwhere they participate in membrane and storage lipid biogenesis. Inchloroplasts, the export process has two steps: firstly, acyl chains arereleased as free fatty acids by the enzymatic activity of acyl-ACPthioesterases (fatty acyl thioesterase; FAT), secondly by reaction withCoA to form acyl-CoA esters which is catalysed by long chain acyl-CoAsynthetases (LACS). A. thaliana contains 3 fatty acyl thioesteraseswhich can be distinguished based on their acyl chain specificity. FATA1and FATA2 preferentially hydrolyze unsaturated acyl-ACPs while saturatedacyl-ACP chains are typically cleaved by FATB.

To explore the effect upon total fatty acid content, TAG content, andfatty acid composition of the co-expression of a thioesterase and genesencoding the WRI1 and/or DGAT polypeptides, chimeric genes were made foreach of the three A. thaliana thioesterases by insertion of the codingregions into the pJP3343 binary expression vector for transientexpression in N. benthamiana leaf cells from the 35S promoter. Proteincoding regions for the A. thaliana FATA1 (Accession No. NP_189147.1, SEQID NO:202) and FATA2 (Accession No. NP_193041.1, SEQ ID NO:203)thioesterases were amplified from silique cDNA using primers containingEcoRI and PstI sites and subsequently cloned into pJP3343 using the samerestriction sites. The resulting expression vectors were designatedpOIL079 and pOIL080, respectively. The protein coding region of the A.thaliana FATB gene (Accession No. NP_172327.1, SEQ ID NO:204) wasamplified using primers containing NotI and SacI flanking sites andcloned into the corresponding restriction sites of pJP3343, resulting inpOIL081. Constructs pOIL079, pOIL080 and pOIL081 are infiltrated into N.benthamiana leaf tissue, either individually or in combination withconstructs containing the genes for the A. thaliana WRI1 transcriptionfactor (AtWRI1) (pJP3414) and/or DGAT1 acyltransferase (AtDGAT1)(pJP3352). For comparison, chimeric genes encoding the Cocos nuciferaFatB1 (CnFATB1) (pJP3630), C. nucifera FatB2 (CnFATB2) (pJP3629) wereintroduced into N. benthamiana leaf tissue in parallel with theArabidopsis thioesterases, to compare the effect of the FatBpolypeptides having MCFA specificity to the Arabidopsis thioesteraseswhich do not have MCFA specificity. All of the infiltrations included achimeric gene for expression of the p19 silencing suppressor asdescribed in Example 1. The negative control infiltrated only the p19T-DNA.

A synergistic effect was observed between thioesterase expression andWRI1 and/or DGAT over-expression on TAG levels in N. benthamiana leaves.Expression of the thioesterase genes without the WRI1 or DGAT genessignificantly increased TAG levels above the low level in the negativecontrol (p19 alone). For example, expression of the coconut FATB2thioesterase resulted in an 8.2-fold increase in TAG levels in theleaves compared to the negative control. Co-expression of the A.thaliana WRI1 transcription factor with each of the thioesterasesfurther increased TAG levels compared to the AtWRI1 control.Co-expression of each of the coconut thioesterases CnFATB1 and CnFATB2with WRI1 resulted in higher TAG levels than each of the three A.thaliana thioesterases with WRI1. Interestingly, the converse wasobserved when the A. thaliana DGAT1 acyltransferase was co-expressed incombination with a thioesterase and WRI1. This suggested a better matchin acyl-chain specificity of the A. thaliana thioesterases and the A.thaliana DGAT1 acyltransferase, resulting in a greater flux ofacyl-chains from the acyl-ACP into TAG. The non-MCFA thioesterases werealso considerably more effective in elevating the percentage of oleicacid in the total fatty acid content in the leaves. Co-expression of theAtWRI1, AtDGAT1 and AtFATA2 resulted in the greatest level of TAG in theleaves, providing a level which was 1.6-fold greater than when AtWRI1and AtDGAT1 were co-expressed without the thioesterase. Theseexperiments confirmed the synergistic increase in oil synthesis andaccumulation when both WRI1 and DGAT were co-expressed as well asshowing the further synergistic increase obtained by adding athioesterase to the combination.

Three different binary expression vectors were constructed to test theeffect of co-expression of genes encoding WRI1, DGAT1 and FATA on TAGlevels and fatty acid composition in stably transformed N. tabacumleaves. The vector pOIL121 contained an SSU::AtWRI1 gene for expressionof AtWRI1 from the SSU promoter, a 35S::AtDGAT1 gene for expression ofAtDGAT from the 35S promoter, and an enTCUP2::AtFATA2 gene forexpression of AtFATA2 from the enTCUP2 promoter which is a constitutivepromoter. These genetic constructs were derived from pOIL38 by firstdigesting the DNA with NotI to remove the gene coding for the S. indicumoleosin. The protein coding region of the A. thaliana FATA2 gene wasamplified and flanked with NotI sites using pOIL80 DNA as template. Thisfragment was then inserted into the NotI site of pOIL38. pOIL121 thenserved as a parent vector for pOIL122 which contained an additionalenTCUP2::SDP1 hairpin RNA cassette for RNAi-mediated silencing of theendogenous SDP1 gene in the transgenic plants. To do this, the entire N.benthamiana SDP1 hairpin cassette was isolated from pOIL51 (Example 11)as an SfoI-SmaI fragment and cloned into the SfoI site of pOIL121,producing pOIL122 (FIG. 3). A third vector, pOIL123, containing theSSU::WRI1 and 35S::DGAT1 genes and the enTCUP2::SDP1 hairpin RNA genewas obtained in a similar way by cloning the enTCUP2::SDP1 hairpin RNAcassette as a SfoI-SmaI fragment into the SfoI site of pOIL36.

In summary, the vectors contained the gene combinations:

pOIL121: SSU::AtWRI1, 35S::AtDGAT1, enTCUP2::AtFATA2.

pOIL122: SSU::AtWRI1, 35S::AtDGAT1, enTCUP2::AtFATA2, enTCUP2::SDP1hairpin.

pOIL123: SSU::AtWRI1, 35S::AtDGAT1, enTCUP2::SDP1 hairpin.

The three constructs were each used to produce transformed N. tabacumplants (cultivar Wi38) by Agrobacterium-mediated transformation.Co-expression of the A. thaliana FATA2 thioesterase or silencing of theendogenous SDP1 TAG lipase in combination with AtWRI1 and AtDGAT1expression each resulted in further elevated TAG levels compared toexpression of AtWRI1 and AtDGAT1 in the absence of both of thethioesterase gene and the SDP1-silencing gene. The greatest TAG yieldswere obtained using pOIL122 by the combined action of all four chimericgenes.

It is noted that N. benthamiana is an 18:3 plant. The same constructspOIL079, pOIL080 and pOIL081 are used to transform A. thaliana, a 16:3plant.

The inventors conceived of the model that increasing plastidial fattyacid export such as by increased fatty acyl thioesterase activityreduces acyl-ACP accumulation in the plastids, thereby increasing fattyacid biosynthesis as a result of reduced feedback inhibition on theacetyl-CoA carboxylase (ACCase) (Andre et al., 2012; Moreno-Perez etal., 2012). Thioesterase over-expression increases export of acyl chainsfrom the plastids into the ER, thereby providing an efficient linkbetween so-called ‘Push’ and ‘Pull’ metabolic engineering strategies.

Example 9. Medium-Chain Fatty Acid Production in Vegetative Plant Cells

Eccleston et al. (1996) studied the accumulation of C12:0 and C14:0fatty acids in both seeds and leaves of transgenic Brassica napus plantstransformed with a constitutively expressed gene encoding California BayLaurel 12:0-ACP thioesterase (Umbellularia californica). That studyreported that substantial levels of C12:0 accumulated in mature B. napusseeds but only very low levels of C12:0 were observed in leaf tissue,despite high levels of 12:0-ACP thioesterase expression and activity.The same results were obtained when the gene was transformed into A.thaliana (Voelker et al., 1992). That research was extended by theco-expression of the Cocos nucifera LPAAT and Umbellularia californicathioesterase which resulted in an increased accumulation of total C12:0as well as an increased fraction of trilaurin in the seeds of B. napus(Knutzon et al., 1999). The prior art therefore indicated that mediumchain fatty acids (MCFA) synthesis in vegetative plant cells wasproblematic.

To test the effect of introducing thioesterases having specificity forMCFA in combination with other genes described herein, chimeric DNAs forexpressing several different thioesterases were synthesized andintroduced into plant cells either singly or in combinations. Theprotein coding regions for thioesterases from organisms known to produceMCFAs (Jing et al., 2011) were synthesised and inserted as EcoRIfragments into the binary vector pJP3343 which contained a 35S-promoterexpression cassette (Vanhercke et al., 2013). The thioesterases were:Cinnamomum camphora 14:0-ACP thioesterase (referred to as Cinca-TE)(Yuan et al., 1995; Accession No. Q39473.1; SEQ ID NO: 193), Cocosnucifera acyl-ACP thioesterase FatB1 (Cocnu-TE1; Accession No.AEM72519.1 SEQ ID NO: 194), Cocos nucifera acyl-ACP thioesterase FatB2(Cocnu-TE2; Accession No. AEM72520.1; SEQ ID NO: 195), Cocos nuciferaacyl-ACP thioesterase FatB3 (Cocnu-TE3; Accession No. AEM72521.1; SEQ IDNO: 196), Cuphea lanceolata acyl-(ACP) thioesterase type B (Cupla-TE)(Topfer et al., 1995; Accession No. CAB60830.1; SEQ ID NO: 197), Cupheaviscosissima FatB1 (Cupvi-TE; Accession No. AEM72522.1; SEQ ID NO: 198)and Umbellularia californica 12:0-ACP thioesterase (Umbca-TE) (Voelkeret al., 1992; Accession No. Q41635.1; SEQ ID NO: 199). Thesethioesterases were all in the FATB class and had specificity for MCFA.The protein coding regions for C. nucifera LPAAT (Cocnu-LPAAT, MCFAtype) (Knutzon et al., 1995; Accession No. Q42670.1; SEQ ID NO: 200) andA. thaliana plastidial LPAAT1 (Arath-PLPAAT; Accession No. AEE85783.1;SEQ ID NO: 201), were also cloned. Cocnu-LPAAT had previously been shownto increase MCFA incorporation on the sn-2 position of TAG in seeds(Knutzon et al., 1995) whilst A. thaliana plastidial LPAAT(Arath-PLPAAT) (Kim et al., 2004) was used as a control LPAAT todetermine the effect of any MCFA specificity that the Cocnu-LPAAT mighthave. The former LPAAT uses acyl-CoA as one substrate and operates inthe ER in its native context, whereas the latter PLPAAT uses acyl-ACP assubstrate and works in the plastid.

The thioesterase genes were introduced into Nicotiana benthamiana leavesby Agrobacterium-mediated infiltration as described in Example 1 alongwith the gene for co-expression of the p19 silencing suppressor andeither the Cocnu-LPAAT or Arath-PLPAAT to determine whether MCFA couldbe produced in N. benthamiana leaf tissue. Infiltrated leaf zones wereharvested and freeze-dried five days after infiltration with theAgrobacterium mixtures, after which the total fatty acid content andcomposition were determined by GC as described in Example 1 (Table 7).For the data shown in Table 7, errors are the standard deviation oftriplicate infiltrations. The infiltrated zones of control leavescontained only trace (<0.1%) or zero levels of fatty acids C12:0 andC14:0 whereas C16:0 was present at 14.9%0±0.6 of the TFA in the totalleaf lipids. C12:0 levels were only increased significantly byexpression of the Cocnu-TE3 (1.2%±0.1) and Umbca-TE (1.6%±0.1).Expression of each of the tested thioesterases resulted in theaccumulation of C14:0 in the N. benthamiana leaves, with Cinca-TE givingthe highest level of 11.3%±1.0. Similarly, expression of each of thethioesterases with the exception of Umbea-TE resulted in increased C16:0levels. The highest level of C16:0 accumulation (35.4%±4.7) was observedwith expression of Cocnu-TE1. Substantial necrosis of the infiltratedzones was observed in the leaves when the FATB genes were expressedalone, which appeared to correlate with the level of MCFA production.The inventors considered that the necrosis was probably due to levels offree fatty acids (FFA) greater than optimum, and also due to theextensive accumulation of MCFA in phospholipid lipid pools rather thanin TAG.

TABLE 7 Total leaf fatty acid composition (% total leaf fatty acid) ofselected fatty acids in Nicotiana benthamiana leaves infiltrated withvarious thioesterases (TE) and LPAATs. Results are grouped by theco-infiltrated gene (single genes (other than p19 present in allsamples), Arath-LPAAT + various TE, Cocnu-LPAAT + various TE). ‘Control’denotes uninfiltrated N. benthamiana leaf whereas ‘p19 only’ containsthe silencing suppressor gene alone. 16:3 is 16:3^(Δ7,10,13); 18:3 is18:3^(Δ9,12,15). Gene identities are defined in the text. 12:0 14:0 16:016:3 18:3 Control 0.2 ± 0 0.1 ± 0   14.0 ± 0.2 8.1 ± 0.1 57.2 ± 0 p19only 0.2 ± 0 0.1 ± 0   14.9 ± 0.6 7.0 ± 0.8 53.1 ± 0.7 Single-gene testsCinca-TE 0.4 ± 0 11.3 ± 1.0  21.9 ± 0.7 5.0 ± 0.2 38.5 ± 1.0 Cocnu-TE10.2 ± 0 6.3 ± 0.6 35.4 ± 4.7 4.2 ± 1.4 29.9 ± 5.5 Cocnu-TE2 0.2 ± 0 7.1± 0.3 31.9 ± 2.2 4.7 ± 0.5 32.9 ± 2.8 Cocnu-TE3 1.2 ± 0.1 7.2 ± 1.3 19.6± 1.6 5.7 ± 0.5 44.8 ± 2.9 Cupla-TE 0.2 ± 0 1.1 ± 0.2 21.8 ± 2.9 6.0 ±0.6 48.2 ± 3.1 Cupvi-TE 0.2 ± 0 0.6 ± 0.1 17.3 ± 1.3 6.4 ± 0.4 52.9 ±2.1 Umbca-TE 1.6 ± 0.1 1.1 ± 0.2 14.4 ± 0.8 6.5 ± 0.3 52.7 ± 0.1 Arath-0.2 ± 0 0.4 ± 0.5 17.4 ± 1.0 6.2 ± 0.3 51.4 ± 1.3 LPAAT Cocnu- 0.1 ± 0.10.1 ± 0   15.1 ± 1.5 6.7 ± 0.5 52.2 ± 4.2 LPAAT +Arath-LPAAT Cinca-TE0.2 ± 0 7.8 ± 0.1 24.6 ± 0.4 5.3 ± 0.2 39.2 ± 1.5 Cocnu-TE1 0.2 ± 0 4.6± 1.3 35.3 ± 1.4 4.4 ± 0.7 32.7 ± 2.0 Cocnu-TE2 0.2 ± 0 6.1 ± 0.4 32.5 ±1.8 4.7 ± 0.1 34.1 ± 0.6 Cocnu-TE3 0.9 ± 0.2 8.5 ± 0.4 21.4 ± 1.9 5.6 ±0.2 41.7 ± 0.6 Cupla-TE 0.2 ± 0 1.0 ± 0.1 23.4 ± 2.7 5.9 ± 0.5 47.3 ±1.2 Cupvi-TE 0.2 ± 0 0.6 ± 0   19.0 ± 0.2 6.3 ± 0.1 51.4 ± 1.0 Umbca-TE1.2 ± 0.2 1.1 ± 0.1 15.4 ± 0.2 6.5 ± 0.2 52.3 ± 1.3 +Cocnu-LPAATCinca-TE 0.7 ± 0.2 14.9 ± 1.6  23.0 ± 3.7 4.8 ± 1.4 35.4 ± 3.3 Cocnu-TE10.2 ± 0 5.4 ± 0.9 40.2 ± 2.8 3.3 ± 0 27.8 ± 1.1 Cocnu-TE2 0.2 ± 0 6.6 ±1.0 38.3 ± 1.1 3.7 ± 0.2 28.2 ± 1.1 Cocnu-TE3 2.0 ± 0.3 10.9 ± 1.0  24.4± 1.8 4.9 ± 0.5 37.7 ± 0.9 Cupla-TE 0.5 ± 0.1 1.6 ± 0.3 22.2 ± 0.6 6.0 ±0.3 46.9 ± 2.0 Cupvi-TE 0.5 ± 0 1.1 ± 0   19.6 ± 0.8 6.0 ± 0.2 49.8 ±0.3 Umbca-TE 3.3 ± 0.5 1.2 ± 0.1 13.9 ± 0.4 6.4 ± 0.2 51.3 ± 1.7

Co-infiltration of the chimeric gene for expressing Arath-PLPAAT withthe thioesterases tended to reduce the accumulation of both C12:0 andC14:0 compared to the absence of the LPAAT, whilst slightly increasingthe accumulation of C16:0. In contrast, co-infiltration of the genes forexpressing Cocnu-LPAAT or Umbca-TE increased the accumulation of C12:0to 3.3%±0.5 whilst C14:0 was found to accumulate to 14.9%±1.6 in theCinca-TE+Cocnu-LPAAT sample. The highest C16:0 levels were observedafter co-expression of Cocnu-TE1 and Cocnu-LPAAT (40.2%±2.8). Additionof an LPAAT to each inoculated zone decreased the degree of necrosis ofthe leaf tissue. Surprisingly, both C8:0 and C10:0 fatty acids were alsoproduced in the plant cells in the transient expression studies. Theaccumulation of C8:0 and C10:0 was not observed when the thioesterasewas expressed alone. However, when thioesterase expression was combinedwith the co-expression of CuphoFatB with CnLPAAT and AtWRI1, C8:0 wasfound to be present at a concentration of 0.27±0.09% of the total fattyacid content in the plant cells. Similarly, when CuplaFatB wasco-expressed with CnLPAAT and AtWRI1, C10:0 was found to be present at0.54±0.16% of the total fatty acid content.

These results indicated that the previously-reported acyl specificitiesof the thioesterases, observed from seed expression, were essentiallymaintained in N. benthamiana leaves and that this expression system wasa valid system for testing acyl specificity. The addition of theplastidial A. thaliana PLPAAT did not increase the accumulation of MCFAsalthough it did result in slightly increased accumulation of C16:0 in A.thaliana cells. In contrast, the C. nucifera LPAAT increased theaccumulation of C12:0, C14:0 and C16:0 in N. benthamiana leaves, whichfatty acids are found in C. nucifera oil (Laureles et al., 2002). Thisindicated that the native N. benthamiana LPAAT was either not highlyexpressed in leaf tissue or did not have high activity on C12:0, C14:0and C16:0 substrates.

Medium-Chain Fatty Acid Production in Vegetative Plant CellsAccumulating High Levels of TAG

The inventors previously obtained the production of 15% TAG in N.tabacum leaves by the coordinate expression of chimeric genes encodingA. thaliana WRI1, A. thaliana DGAT1 and S. indicum Oleosin (Vanhercke etal., 2014). To test whether the accumulation of MCFA that was observedafter expression of thioesterases in combination with an LPAAT wouldalso occur or be increased in plant cells producing high levels of TAG(Vanhercke et al., 2013), these genes were co-expressed. The bestperforming C12:0, C14:0 and C16:0 thioesterase/LPAAT combinations(Cocnu-LPAAT plus Umbca-TE, Cinca-TE and Cocnu-TE2 thioesterases,respectively) were infiltrated with and without the Arath-WRI1+DGATcombinations previously described (Vanhercke et al., 2013). The data areshown in FIG. 4.

The accumulation of the relevant MCFA (C12:0 for Umbca-TE, C14:0 forCinca-TE and C16:0 for Cocnu-TE2) was consistently and substantiallyincreased most by the addition of Arath-WRI1 to the combinations: C12:0comprised 9.5%±0.9 of total leaf fatty acids in theUmbca-TE+Cocnu-LPAAT+Arath-WRI1 samples, the C14:0 level was 18.5%±2.6in the Cinca-TE+Cocnu-LPAAT+Arath-WRI1 samples and the C16:0 level was38.3%±3.0 in the Cocnu-TE2+Cocnu-LPAAT+Arath-WRI1 samples. Thioesteraseplus Arath-WRI1 infiltrations were found to have a significantly greatereffect on C12:0 in the presence of Umbca-TE, C14:0 in the presence ofCinca-TE and C16:0 in the presence of Cocnu-TE2 relative to infiltrationwith thioesterase plus Cocnu-LPAAT in the absence of WRI1 (FIG. 5). Theaddition of the Cocnu-LPAAT to the thioesterase plus Arath-WRI1 mixturesdid have an effect on the fatty acid composition with relatively smallincreases in C12:0 and C14:0 observed in the Umbca-TE and Cinca-TE setsand a small decrease in C16:0 in the Cocnu-TE2 set. The maximum levelsobserved were: 8.8%±1.1 of C12:0 in total leaf fatty acids observed inthe Umbca-TE+Arath-WRI1+Cocnu-LPAAT samples, 14.1%±3.5 of C14:0 in theCinca-TE+Arath-WRI1+Cocnu-LPAAT samples and 48.6%±3.7 of C16:0 in theCocnu-TE2+Arath-WRI1 sample.

Interestingly, the only thioesterase in which the Arath-WRI1 did notincrease MCFA accumulation as much was the Cocnu-TE2, although it stillincreased significantly. The addition of this gene alone resulted in theincreased accumulation of C16:0 from 16.0%-0.4 to 37.3%±0.6 whereas thefurther addition of Arath-WRI1 only increased this to 48.6%±1.7. Thismay have been due to the C12:0 and C14:0 intermediates being relativelytransient during plastidial fatty acid synthesis compared to C16:0.

Other effects that were noted included the increase in C16:0 andC18:1^(Δ9) and decrease in C18:3^(Δ9,12,15) levels in the presence ofArath-WRI1. The further addition of the Cinca-TE and Cocnu-TE2 decreasedC18:3^(Δ9,12,15) levels further still. In contrast, the extra C12:0produced following the addition of Arath-WRI1 to Umbca-TE appeared tocome at the cost of C16:0 rather than additional C18:3^(Δ9,12,15) (FIG.5).

A subset of samples were also analysed by LC-MS to gain a betterunderstanding of MCFA accumulation. The plastidial galactolipidsmonogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol(DGDG) contained only low levels of C12:0 and C14:0 and reduced levelsof C16:0 relative to the p19 control infiltration. The majorC12:0-containing MGDG species in the Umbca-TE samples was 30:3indicating that one C18:3 and one C12:0 were co-located on themonogalactosyl backbone. The other main C12:0-containing MGDG specieswas 28:0, indicating that the second fatty acid was C16:0. The majorC14:0-containing MGDG species in the Cinca-TE samples were 28:0 and30:0, indicating that a significant proportion of the C14:0 in MGDG waseither di-C14:0 or with C16:0. The C12:0-containing and C14:0-containingMGDG species were not detected in the p19 control sample. In contrast,C16:0-containing MGDG species tended to be reduced in the Cocnu-TE2samples. The major MGDG species in the wildtype samples(C16:3-containing 34:6, C18:3-containing 34:6, and C18:3-containing36:6) all tended to be reduced by the expression of the transgenes. Thisreduction was greatest in the presence of the WRI+DGAT combination.

Only trace levels of C12:0-containing DGDG species were observed in theUmbca-TE samples. The major C14:0-containing species observed in theCinca-TE samples were 28:0 and 30:0, both of which were absent in thecontrol. These species were also observed at elevated levels in theCocnu-TE2 samples but only at trace levels in the Umbca-TE samples. Themajor DGDG species in the wildtype samples (C16:0-containing 34:3,C18:3-containing 34:3, and C18:3-containing 36:6) all tended to bereduced by the expression of the transgenes. This reduction was greatestin the presence of WRI.

Similarly, TAG species were generally increased considerably in all thesamples containing WRI+DGAT as previously described (Vanhercke et al.,2013). C12:0 species were found to be dominant in the high TAG Umbca-TEsample, C14:0 in the high TAG Cinca-TE sample and C16:0 in the high TAGCocnu-TE2 sample. LC-MS analysis of the TAG fraction showed that theC12:0-containing 36:0 was found to be the dominant TAG species, twicethe level of TAG species containing C18:3, in all Umbca-TE samplescontaining the WRI1 transcription factor. Similarly, C14:0-containing42:0 was the dominant TAG species in the Cinca-TE samples co-transformedwith either LPAAT, DGAT, WRI1 or WRI+DGAT, although the response wasconsiderably higher in the case of the samples containing WRI. SeveralC16:0-containing TAG species were significantly elevated in both thehigh TAG Cinca-TE (e.g. 44:0 and 50:3) and Cocnu-TE2 (e.g. 46:0, 48:0,50:2 and 50:3) samples. Again, the greatest C16:0 increases wereobserved in the presence of WRI.

Stable Transformation for Production of MCFA in Vegetative Tissues.

A series of genetic constructs were made in a binary vector in order tostably transform plants such as tobacco with combinations of genes forproduction of MCFA in vegetative tissues, to identify optimalcombinations of genes. These constructs included a gene for expressionof WRI1 under the control of either the SSU promoter (see Example 8,pOIL121) or the senescence-specific SAG12 promoter, a gene encoding anoil palm DGAT (below), a gene encoding the coconut LPAAT (CocnuLPAAT,see above) under the control of an enTCUP promoter and several genesexpressing a variety of fatty acyl thioesterases (FATB) expressed fromeither a 35S promoter or a SAG12 promoter. These are described below.

Cloning of a Gene Encoding Elaeis guineensis (Oil Palm) DGAT

In order to firstly test different DGAT enzymes, includingrepresentative DGAT1, DGAT2 and DGAT3 enzymes, candidate oil palm DGATsequences were identified from the published transcriptome (Dussert etal., 2013) and codon optimised for expression in Nicotiana tabacum. Theprotein coding regions were then each cloned individually into binaryexpression vectors under the control of the 35S promoter for testing intransient N. benthamiana leaf assays as described in Example 1. The genecombinations tested were as follows:

1 P19 (negative control)

2 P19+CnLPAAT+WRI1

3 P19+CnLPAAT+AtWRI1+AtDGAT1

4 P9+CnLPAAT+AtWRI1+EgDGAT1

5 P19+CnLPAAT+AtWRI1+EgDGAT2

6 P19+CnLPAAT+AtWRI1+EgDGAT3

7 P19+CincaFatB

8 P19+CincaFatB+CnLPAAT+WRI1

9 P19+CincaFatB+CnLPAAT+AtWRI1+AtDGAT1

10 P19+CincaFatB+CnLPAAT+AtWRI1+EgDGAT1

11 P19+CincaFatB+CnLPAAT+AtWRI1+EgDGAT2

12 P19+CincaFatB+CnLPAAT+AtWRI1+EgDGAT3

The results for the TFA and TAG levels, and the levels of total MCFA inthe TFA or the TAG contents, are shown in FIG. 6. Compared to AtDGAT1,the expression of EgDGAT1 led to greater accumulation of total fattyacids and increased TAG levels. The total MCFA content in the totalfatty acid content was reduced with the expression of EgDGAT1 relativeto AtDGAT1, but the levels of MCFA present in TAG remained about thesame (FIG. 6).

Preparation of Genetic Constructs

Genetic constructs for stable transformation (Table 8) were assembledthrough the sequential insertion of gene cassettes through the use ofcompatible restriction enzyme sites. The four gene constructs (Table 8)each contained a gene encoding the oil palm DGAT1 (EgDGAT1) expressedfrom the 35S promoter, a gene encoding the C. nucifera LPAAT (CnLPAAT)expressed from the constitutive enTCUP2 promoter, and a gene encodingAtWRI1 expressed from either the SSU promoter or the SAG12 promoter inaddition to one of a series of genes encoding FATB enzymes.

The five gene constructs also contained a gene for expression of ahairpin RNA for reducing expression of an endogenous gene encodingacyl-activating enzyme (AAE). The hairpin was constructed based onsequence similarity with the identified AAE15 from Arabidopsis lyrata(EFH44575.1) and the N. benthamiana genome. AAE has been shown to beinvolved in the reactivation of MCFA, and hence further elongation. Itwas considered that silencing of AAE might increase MCFA accumulation.The hairpin cassette was constructed in the vector pKANNIBAL and thensubcloned into the expression vector pWBVec2 (Wang et al., 2004), withthe expression of the hairpin being driven by the 35S promoter.

TABLE 8 Summary of assembled genetic constructs. Construct GeneCombination Single Gene pKR1 35S::UmbcaFATB Constructs pKR235S::CincaFATB pKR3 35S::CocnuFATB2 pOIL115 SAG12::CincaFATB pOIL116SAG12::UmbcaFATB pOIL117 SAG12::CocnuFATB2 Construction pOIL30035S::EgDGAT1 Components pOIL301 enTCUP::CnLPAAT inFATBrmediaFATBconstruct pOIL302 35S::EgDGAT1 + enTCUP::CnLPAAT pOIL303 35S::EgDGAT1 +enTCUP::CnLPAAT + SSU:AtWRI1 pOIL304 35S::EgDGAT1 + enTCUP::CnLPAAT +SAG12:AtWRI1 Four Gene Constructs pOIL305 35S::EgDGAT1 +enTCUP::CnLPAAT + SSU:AtWRI1 + 35S::UmbcaFATB pOIL306 35S::EgDGAT1 +enTCUP::CnLPAAT + SSU:AtWRI1 + 35S::CincaFATB pOIL307 35S::EgDGAT1 +enTCUP::CnLPAAT + SSU:AtWRI1 + 35S::CocnuFATB2 pOIL308 35S::EgDGAT1 +enTCUP::CnLPAAT + SSU:AtWRI1 + SAG12::UmbcaFATB pOIL309 35S::EgDGAT1 +enTCUP::CnLPAAT + SSU:AtWRI1 + SAG12::CincaFATB pOIL310 35S::EgDGAT1 +enTCUP::CnLPAAT + SSU:AtWRI1 + SAG12::CocnuFATB2 pOIL311 35S::EgDGAT1 +enTCUP::CnLPAAT + SAG12:AtWRI1 + 35S::UmbcaFATB pOIL312 35S::EgDGAT1 +enTCUP::CnLPAAT + SAG12:AtWRI1 + 35S::CincaFATB pOIL313 35S::EgDGAT1 +enTCUP::CnLPAAT + SAG12:AtWRI1 + 35S::CocnuFATB2 pOIL314 35S::EgDGAT1 +enTCUP::CnLPAAT + SAG12:AtWRI1 + SAG12::UmbcaFATB pOIL315 35S::EgDGAT1 +enTCUP::CnLPAAT + SAG12:AtWRI1 + SAG12::CincaFATB pOIL316 35S::EgDGAT1 +enTCUP::CnLPAAT + SAG12:AtWRI1 + SAG12::CocnuFATB2 Five Gene ConstructspOIL317 35S::EgDGAT1 + enTCUP::CnLPAAT + SSU:AtWRI1 + 35S::UmbcaFATB +35S::hpNbAAE pOIL318 35S::EgDGAT1 + enTCUP::CnLPAAT + SSU:AtWRI1 +35S::CincaFATB + 35S::hpNbAAE pOIL319 35S::EgDGAT1 + enTCUP::CnLPAAT +SSU:AtWRI1 + 35S::CocnuFATB2 + 35S::hpNbAAE pOIL320 35S::EgDGAT1 +enTCUP::CnLPAAT + SSU:AtWRI1 + SAG12::UmbcaFATB + 35S::hpNbAAE pOIL32135S::EgDGAT1 + enTCUP::CnLPAAT + SSU:AtWRI1 + SAG12::CincaFATB +35S::hpNbAAE pOIL322 35S::EgDGAT1 + enTCUP::CnLPAAT + SSU:AtWRI1 +SAG12::CocnuFATB2 + 35S::hpNbAAE pOIL323 35S::EgDGAT1 +enTCUP::CnLPAAT + SAG12:AtWRI1 + 35S::UmbcaFATB + 35S::hpNbAAE pOIL32435S::EgDGAT1 + enTCUP::CnLPAAT + SAG12:AtWRI1 + 35S::CincaFATB +35S::hpNbAAE pOIL325 35S::EgDGAT1 + enTCUP::CnLPAAT + SAG12:AtWRI1 +35S::CocnuFATB2 + 35S::hpNbAAE pOIL326 35S::EgDGAT1 + enTCUP::CnLPAAT +SAG12:AtWRI1 + SAG12::UmbcaFATB + 35S::hpNbAAE pOIL327 35S::EgDGAT1 +enTCUP::CnLPAAT + SAG12:AtWRI1 + SAG12::CincaFATB + 35S::hpNbAAE pOIL32835S::EgDGAT1 + enTCUP::CnLPAAT + SAG12:AtWRI1 + SAG12::CocnuFATB2 +35S::hpNbAAE

These genetic constructs were used to produce transformed tobacco plantsof cultivars Wisconsin 38 and a high oil line transformed with the T-DNAfrom pJP3502. It was observed that plants transformed with the singlegene FATB constructs expressed from the 35S promoter were significantlysmaller than those transformed with the corresponding FATB constructexpressed from the SAG12 promoter or from the four gene constructs. Thesmaller plant size was considered to be caused by a buildup of MCFAwhich was not incorporated efficiently into TAG.

Discussion

The present study found that C12:0 production in leaf cells was onlyabout 1.6% of the total fatty acid content after expression of Umbca-TEalone (Table 7). The addition of a gene for expression of Arath-WRI1 hada much stronger effect on C12:0 and C14:0 accumulation in leaf tissuethan the addition of the coconut LPAAT (FIGS. 4 and 5). This indicatedthat WRI1 in combination with the thioesterase greatly increased MCFAaccumulation in leaf cells, acting synergistically. Importantly, much ofthe C12:0, C14:0 and C16:0 was found to accumulate in the leaves in TAG,which lipid does not accumulate at substantial levels in wild-typeleaves. These experiments showed that the cells in the vegetative partsof plants could be modified to produce MCFA, particularly C12:0 andC14:0 in TAG at high levels. C16:0 levels were also increasedsubstantially.

Example 10. The Effect of Different Transcription Factor Polypeptides onTAG Accumulation

Previously reported experiments with WRI1 and DGAT (Vanhercke et al.,2013) used a synthetic gene encoding A. thaliana AtWRI1 (Accession No.AAP80382.1) and a synthetic gene encoding AtDGAT1, also from A. thaliana(Accession No. AAF19262; SEQ ID NO: 1). To compare other WRI1polypeptides with AtWRI1 for their ability to combine with DGAT toincrease oil content, other WRI coding sequences were identified andused to generate constructs for expression in N. benthamiana leaves.Nucleotide sequences encoding the A. thaliana WRI3 (Accession No.AAM91814.1, SEQ ID NO:205) and WRI4 (Accession No. NP_178088.2, SEQ IDNO:206) transcription factors (To et al., 2012) were synthesized andinserted as EcoRI fragments into pJP3343 under the control of the 35Spromoter. The resulting binary expression vectors were designatedpOIL027 and pOIL028, respectively. The coding sequence for the oat(Avena sativa) WRI1 (AsWRI1, SEQ ID NO:207) was PCR amplified from avector provided by Prof. Sten Stymne (Swedish University of AgriculturalSciences) using flanking primers containing additional EcoRI sites. Theamplified fragment was inserted into pJP3343 resulting in pOIL055. AWRI1 candidate sequence from S. bicolor (Accession No. XP_002450194.1,SEQ ID NO:208) was identified by a BLASTp search on the NCBI serverusing the Zea mays WRI1 amino acid sequence (Accession No.NP_001137064.1, SEQ ID NO:209) as query. The protein coding region ofthe S. bicolor WRI) gene (SbWRI1) was synthesized and inserted as anEcoRI fragment into pJP3343, yielding pOIL056. A gene candidate encodinga WRI1 was identified from the Chinese tallow (Triadica sebifera;TsWRI1, SEQ ID NO:210) transcriptome (Uday et al., submitted). Theprotein coding region was synthesized and inserted as an EcoRI fragmentinto pJP3343 resulting in pOIL070. The pJP3414 and pJP3352 binaryvectors containing the coding sequences for expression of the A.thaliana WRI1 and DGAT1 polypeptides were as described by Vanhercke etal. (2013).

Plasmids containing the various WRI1 coding sequences were introducedinto N. benthamiana leaf tissue for transient expression using a geneencoding the p19 viral suppressor protein in all inoculations asdescribed in Example 1. The genes encoding the WRI1 polypeptides wereeither tested alone or in combination with the DGAT1 acyltransferasegene, the latter to provide greater TAG biosynthesis and accumulation.The positive control in this experiment was the combination of the genesencoding A. thaliana WRI1 transcription factor and AtDGAT1. Allinfiltrations were done in triplicate using three different plants andTAG levels were analyzed as described in Example 1. Expression of mostof the individual WRI1 polypeptides in the absence of exogenously addedDGAT1 resulted in increased, yet still low, TAG levels (<0.23% on dryweight basis) in infiltrated leaf spots, compared to the control whichhad only the p19 construct (FIG. 7). The exception was TsWRI1 which, byitself, did not appear to increase TAG levels significantly. Inaddition, differences in TAG levels produced by expression of thedifferent WRI1 transcription factors on their own were not great. BothAsWRI1 and SbWRI1 yielded TAG levels similar to AtWRI1 on its own.Analysis of the TAG fatty acid composition revealed only minor changesexcept for increased C18:1Δ9 levels from expression of AtWRI3 in theinfiltrated leaf tissues (Table 9).

TABLE 9 TAG fatty acid composition in N. benthamiana leaf samplesinfiltrated with different chimeric genes for expression of WRI (n = 3).All samples were also infiltrated with the P19 construct. The TAGsamples also contained 0.1-0.4% C14:0; 0.5-1.2% C16:3 and; 0.1-0.7%C18:1Δ11. Infiltrated genes C16:0 C16:1 C18:0 C18:1 C18:2 C18:3n3 C20:0C20:1 C22:0 C24:0 Control (P19) 33.6 ± 4.7 0.5 ± 0.4 8.9 ± 2.2 4.7 ± 0.616.9 ± 1.0 32.2 ± 7.8 1.1 ± 0.2 0.8 ± 1.5 0.0 0.0 WRI1 35.5 ± 3.4 0.7 ±0.2 5.2 ± 0.8 5.4 ± 1.3 17.1 ± 1.0 33.1 ± 2.7 0.8 ± 0.1 0.5 ± 0.6 0.3 ±0.0 0.0 WRI3 27.3 ± 1.6 0.9 ± 0.2 4.8 ± 0.3 10.2 ± 1.5  16.1 ± 1.0 37.8± 1.2 0.8 ± 0.1 0.6 ± 0.7 0.1 ± 0.2 0.0 WRI4 30.1 ± 0.4 1.0 ± 0.4 5.2 ±0.8 4.6 ± 0.6 17.2 ± 0.4 38.1 ± 1.6 0.8 ± 0.1 1.3 ± 1.3 0.0 0.0 AsWRI35.7 ± 3.0 1.7 ± 0.4 5.3 ± 0.7 6.5 ± 0.3 15.4 ± 0.4 31.6 ± 1.6 0.8 ± 0.10.4 ± 0.7 0.3 ± 0.1 0.0 SbWRI 37.4 ± 0.8 1.9 ± 0.3 4.8 ± 0.3 7.0 ± 1.215.2 ± 0.3 30.8 ± 0.3 0.8 ± 0.1 0.4 ± 0.6 0.3 ± 0.0 0.0 TsWRI 34.5 ± 4.80.0 9.4 ± 8.2 5.9 ± 1.7 16.0 ± 0.7  29.3 ± 12.4 0.0 n.d. 0.0 0.0 Control(P19) 31.0 ± 2.1 0.9 ± 0.1 8.7 ± 1.3 8.0 ± 2.3 24.9 ± 1.5 22.1 ± 4.7 2.0± 0.1 0.0 0.6 ± 0.6 0.2 ± 0.4 WRI1 + DGAT 27.7 ± 0.1 0.3 ± 0.0 7.0 ± 0.117.2 ± 0.7  27.9 ± 0.9 14.7 ± 0.3 2.4 ± 0.2 0.3 ± 0.0 1.1 ± 0.1 0.8 ±0.2 WRI3 + DGAT 30.0 ± 0.8 0.6 ± 0.1 5.9 ± 0.4 13.9 ± 2.9  21.5 ± 1.121.3 ± 0.8 2.8 ± 0.1 0.2 ± 0.0 1.8 ± 0.1 1.0 ± 0.2 WRI4 + DGAT 27.0 ±0.5 0.2 ± 0.1 8.5 ± 0.2 5.8 ± 0.7 23.9 ± 0.8 25.2 ± 1.3 3.5 ± 0.1 0.2 ±0.0 2.1 ± 0.2 1.7 ± 0.2 AsWRI + DGAT 33.8 ± 0.5 1.1 ± 0.1 5.5 ± 0.9 12.2± 1.6  26.0 ± 1.9 16.3 ± 1.3 2.2 ± 0.2 0.2 ± 0.0 1.2 ± 0.1 0.8 ± 0.1SbWRI + DGAT 34.6 ± 0.5 1.3 ± 0.1 5.6 ± 0.4 13.9 ± 1.6  23.6 ± 1.3 15.8± 0.6 2.2 ± 0.1 0.2 ± 0.0 1.2 ± 0.1 0.9 ± 0.1 TsWRI + DGAT 25.4 ± 0.50.2 ± 0.0 9.4 ± 0.1 7.7 ± 1.0 27.0 ± 1.3 22.1 ± 2.4 3.6 ± 0.2 0.2 ± 0.01.8 ± 0.2 1.3 ± 0.2

In contrast, differences in TAG yields from expression of the differentWRI polypeptides were more pronounced upon co-expression with theAtDGAT1 acyltransferase. This again demonstrated the synergistic effectof WRI1 and DGAT co-expression on TAG biosynthesis in infiltrated N.benthamiana leaf tissue, as reported by Vanhercke et al. (2013).Intermediate TAG levels were observed upon co-expression of DGAT1 withAtWRI3, AtWRI4 and TsWRI1 expressing vectors while levels obtained withthe AsWRI1 and AtWRI1 were significantly lower. In a result that couldnot have been predicted beforehand, the highest TAG yields were obtainedwith co-expression of DGAT with SbWRI1, even though the assay was donein dicotyledonous cells. TAG fatty acid composition analysis revealedincreased levels of C18:1^(Δ9) and decreased levels of C18:3^(Δ9,12,15)(ALA) in the case of SbWRI1, AsWRI1 and the AtWRI1 positive control(Table 9). Unlike AtWRI1, however, expression of AsWRI1 and SbWRI1 bothdisplayed increased C16:0 levels compared to the p19 negative control.Interestingly, AtWRI3 infiltrated leaf samples exhibited a distinct TAGprofile with C18:1^(Δ9) being enriched while C16:0 and ALA were onlyslightly affected.

This experiment showed that the S. bicolor WRI1 transcription factor,SbWRI1, was superior to AtWRI1 when co-expressed with DGAT to increaseTAG levels in vegetative plant parts. The inventors also concluded thata transcription factor, for example a WRI1, from a monocotyledonousplant could function well in a dicotyledonous plant cell, indeed mighteven have superior activity compared to a corresponding transcriptionfactor from a dicotyledonous plant. Likewise, a transcription factorfrom a dicotyledonous plant could function well in a monocotyledonousplant cell.

Use of Other Transcription Factors

Genetic constructs were prepared for expression of each of 14 differenttranscription factors in plant cells to test their ability to functionfor increasing TAG levels in combination with other genes involved inTAG biosynthesis and accumulation. These transcription factors werecandidates as alternatives for WRI1 or for addition to combinationsincluding one or more of WRI1, LEC1 and LEC2 transcription factors foruse in plant cells, particularly in vegetative plant parts. Theirselection was largely based on their reported involvement inembryogenesis (reviewed in Baud and Lepiniec (2010), and Ikeda et al.(2006)), similar to LEC2. Experiments were therefore carried out toassay their function, using the N. benthamiana expression system(Example 1), as follows.

Nucleotide sequences of the protein coding regions of the followingtranscription factors were codon optimized for expression in N.benthamiana and N. tabacum, synthesized and subcloned as NotI-SacIfragments into the respective sites of pJP3343: A. thaliana FUS3(pOIL164) (Luerssen et al., 1998; Accession number AAC35247; SEQ IDNO:160), A. thaliana LEC1L (pOIL165) (Kwong et al. 2003; Accessionnumber AAN15924; SEQ ID NO:157), A. thaliana LEC1 (pOIL166) (Lotan etal., 1998; Accession number AAC39488; SEQ ID NO:149), G. max MYB73(pOIL167) (Liu et al., 2014; Accession number ABH02868; SEQ ID NO:221),A. thaliana bZIP53 (pOIL168) (Alonso et al., 2009; Accession numberAAM14360; SEQ ID NO:222), A. thaliana AGL15 (pOIL169) (Zheng et al.,2009; Accession number NP_196883; SEQ ID NO:223), A. thaliana MYB118(Accession number AAS58517; pOIL170; SEQ ID NO:224), MYB115 (Wang etal., 2002; Accession number AAS10103; pOIL171; SEQ ID NO:225), A.thaliana TANMEI (pOIL172) (Yamagishi et al., 2005; Accession numberBAE44475; SEQ ID NO:226), A. thaliana WUS (pOIL173) (Laux et al., 1996;Accession number NP_565429; SEQ ID NO:227), A. thaliana BBM (pOIL174)(Boutilier et al., 2002; Accession number AAM33893, SEQ ID NO:145), B.napus GFR2a1 (Accession number AFB74090; pOIL177; SEQ ID NO:228) andGFR2a2 (Accession number AFB74089; pOIL178; SEQ ID NO:229) (Liu et al.(2012c)). In addition, a codon optimized version of the A. thaliana PHR1transcription factor involved in adaptation to high light phosphatestarvation conditions was similarly subcloned into pJP3343 (pOIL189)(Nilsson et al (2012); Accession number AAN72198; SEQ ID NO:230). Thesetranscription factors are summarised in Table 10.

As a screening assay to determine the function of these transcriptionfactors, the genetic constructs are introduced into N. benthamiana leafcells as described in Example 1, either with or without a gene encodingDGAT1, or other gene combinations such as encoding WRI1, LEC2, hpSDP1 orFATA thioesterase, and total lipid content and fatty acid composition ofthe leaf cells is measured. Transcription factors that increased totallipid contents significantly are identified and selected.

For stable transformation of plants using genes encoding the alternativetranscription factors, the following binary constructs are made. Thegenes for expression of the transcription factors use either the SSUpromoter or the SAG12 promoter. Over-expression of embryogenictranscription factors such as LEC1 and LEC2 has been shown to induce avariety of pleotropic effects, undesirable in the present context,including somatic embryogenesis (Feeney et al. (2012); Santos-Mendoza etal. (2005); Stone et al. (2008); Stone et al. (2001); Shen et al.(2010)). To minimize possible negative impact on plant development andbiomass yield, tissue or developmental-stage specific promoters arepreferred over constitutive promoters to drive the ectopic expression ofmaster regulators of embryogenesis.

TABLE 10 Additional transcription factors and the genetic constructs fortheir expression Transcription Length (amino Accession Plasmid factorSpecies acid) number pOIL164 FUS3 A. thaliana 312 AAC35247 pOIL165 LEC1LA. thaliana 234 AAN15924 pOIL166 LEC1 A. thaliana 208 AAC39488 pOIL167MYB73 G. max 74 ABH02868 pOIL168 bZIP53 A. thaliana 146 AAM14360 pOIL169AGL15 A. thaliana 268 NP_196883 pOIL170 MYB118 A. thaliana 437 AAS58517pOIL171 MYB115 A. thaliana 359 AAS10103 pOIL172 TANMEI A. thaliana 386BAE44475 pOIL173 WUS A. thaliana 292 NP_565429 pOIL174 BBM A. thaliana584 AAM33803 pOIL177 GFR2a1 B. napus 453 AFB74090 pOIL178 GFR2a2 B.napus 461 AFB74089 pOIL189 PHR1 A. thaliana 409 AAN72198

Example 11. Silencing of a TAG Lipase in Plants Accumulating High Levelsof TAG in Leaf Tissue

The Sugar Dependent 1 (SDP1) TAG lipase has been demonstrated to play arole in TAG turnover in non-seed tissues of A. thaliana as well asduring seed germination (Eastmond et al., 2006; Kelly et al., 2011;Kelly et al., 2013). SDP1 is expressed in developing seed and the SDP1polypeptide is also present in mature seed in association with (but notcoating) oil bodies. Silencing of the gene encoding SDP1 resulted in asmall but significant increase in TAG levels in A. thaliana roots andstems (<0.4% on dry weight basis) while an even smaller increase wasobserved in leaf tissue (Kelly et al., 2013).

To determine whether TAG levels could be increased further in leaf andstem tissues relative to co-expression of AtWRI1 and AtDGAT1, anexperiment was designed to silence an endogenous SDP1 gene in N. tabacumplants which were homozygous for a T-DNA having genes for transgenicexpression of the WRI, DGAT1 and Oleosin polypeptides (Vanhercke et al.,2014). A BLAST search of the N. benthamiana transcriptome (Naim et al.,2012) using the AtSDP1 nucleotide sequence as query identified atranscript (Nbv5 tr6385200, SEQ ID NO:173) with homology to the A.thaliana SDP1 gene. A 713 bp region (SEQ ID NO:174) was selected forhairpin mediated gene silencing. A 3.903 kb synthetic fragment wasdesigned, based on the pHELLSGATE12 vector, which comprised, in order,the enTCUP2 constitutive promoter, the 713 bp N. benthamiana SDP1fragment in sense orientation flanked by attB1 and attB2 sites, a Pdkintron, a cat intron sequence in reverse orientation, a second 713 bp N.benthamiana SDP1 fragment flanked by attB1 and attB2 sites in reverse(antisense) orientation, and the OCS 3′ regionterminator/polyadenylation site (FIG. 8). The insert was subcloned intopJP3303 using Smina and KasI restriction sites and the resultingexpression vector was designated pOIL051. This chimeric DNA contains ahygromycin resistance selectable marker gene.

pOIL051 was used to produce transformed N. tabacum plants byAgrobacterium-mediated transformation. The starting plant cells werefrom transgenic plants which were homozygous for the T-DNA of pJP3502(Vanhercke et al., 2014). Transgenic plants containing the T-DNA frompOIL051 were selected by hygromycin resistance and transferred to soilin the glasshouse or in a controlled environment cabinet for continuedgrowth. Leaf samples were harvested from confirmed double-transformants(T0 plants) before flowering, at flowering and at seed setting stages ofplant development, and the TAG level in each determined. Transgenicplants containing only low levels of leaf TAG, or TAG at the same levelas controls, were identified by means of lipid extraction from leafsamples and analysis by spot TLC and discarded. TAG levels in theremaining population of transformants were quantified by GC as describedin Example 1. Before flowering, the majority of these plants exhibitedgreatly increased TAG levels (>5% of leaf dry weight) in their leaftissue while 4 plants contained TAG levels above 10% (Table 11). Themaximum TAG level observed in leaves of these plants, before flowering,was 11.3% in plant 51-13. As a comparison, the transgenic plants of theparental N. tabacum line expressing AtWRI1, AtDGAT1 and Oleosindisplayed TAG levels of about 2% before flowering and about 6% duringflowering (Vanhercke et al., 2014). The addition of the SDP1-inhibitoryconstruct to the AtWRI1 plus AtDGAT1 combination was thereforesynergistic for increasing the TAG levels in these plants. Surprisingly,the TAG content in leaves harvested from the doubly-transformed plantsat flowering stage was greatly increased, observing 30.5% on a dryweight basis (Table 12), representing a 5-fold increase relative to theplants not silenced for SDP1. To the great amazement of the inventors,the TAG level reached an astonishing 70.7% (% of dry weight) in samplesof senescing leaves (green and yellow) at the seed setting stage (Table13). When NMR was used to measure the oil content of entire leaves fromthe tobacco plants at seed setting stage, the TAG content in some greenleaves that had started senescing was about 43% and in some brown,desiccated leaves was 42%. When such leaves were pressed between twobrown paper filters, the exuded oil soaked into the paper and made ittranslucent, whereas control tobacco leaves did not do so, providing asimple screening method for detecting plants having high oil content.

Two primary transformants (#61, #69) containing each of the T-DNAs frompJP3502 and pOIL51 and displaying high TAG levels were analyzed bydigital PCR (ddPCR) using a hygromycin gene-specific primer pair todetermine the number of pOIL51 T-DNA insertions. The plant designated#61 contained one T-DNA insertion from pOIL51, whereas plant #69contained three T-DNA insertions from pOIL51. T1 progeny plants of bothlines were screened again by ddPCR to identify homozygous, heterozygousand null plants. Progeny plants of plant #61 containing no insertionsfrom pOIL51 (nulls; total of 7) or 2 T-DNA insertions (i.e. homozygousfor that T-DNA; total of 12) were selected for further analysis.Similarly, progeny plants of line #69 containing zero T-DNA insertionsfrom pOIL51 (nulls; total of 2) or 2 such insertions (total of 15) or 4or 5 insertions (total of 5) were maintained for further analysis.

TABLE 11 TAG levels (% leaf dry weight) and TAG fatty acid compositionin leaf tissue from N. tabacum plants (T0 generation) expressing WRI1,DGAT1 and Oleosin transgenes and super-transformed with a T-DNA encodingan SDP1 hairpin construct (pOIL051), compared to wild-type(untransformed). Leaf samples were harvested during vegetative stage(before flowering). Lipid samples also contained 0.0-0.2% C16:3,0.0-0.4% C20:1; 0.0-0.1% C20:2n-6. Line C14:0 C16:0 C16:1 C18:0 C18:1C18:1d11 C18:2 C18:3n3 C20:0 C22:0 C24:0 % TAG wild- 2.5 20.2 0.0 8.65.6 0.0 18.9 44.2 0.0 0.0 0.0 0.1 type 23-31 0.0 66.0 0.0 0.0 34.0 0.00.0 0.0 0.0 0.0 0.0 0.0 23-29 0.0 36.1 1.4 5.1 21.0 0.8 23.3 7.1 2.4 1.51.1 2.9 57 0.1 47.2 0.3 5.4 19.2 1.9 0.0 21.2 2.1 1.2 1.1 3.4 23-1  0.230.8 1.9 4.9 41.2 1.0 13.7 2.4 1.9 1.1 0.7 4.0 58 0.1 31.4 0.2 3.8 12.21.6 33.6 13.2 1.7 1.0 0.7 4.0 21 0.1 31.9 0.3 3.9 10.7 1.5 32.3 15.2 1.91.1 0.8 4.7 23-30 0.2 34.1 0.7 4.9 29.4 0.9 17.5 5.7 2.9 1.8 1.7 4.9 400.1 34.4 0.2 4.3 14.3 1.5 29.7 11.8 1.7 1.0 0.7 5.1 22 0.1 35.8 0.2 4.312.8 1.5 29.8 11.7 1.8 1.0 0.7 5.1 15 0.1 37.2 0.1 3.9 8.6 1.7 29.3 16.01.5 0.8 0.6 5.1 16 0.1 35.2 0.1 3.9 13.9 1.7 28.5 13.6 1.4 0.7 0.6 5.325 0.1 34.4 0.2 3.9 15.4 1.8 27.6 13.2 1.6 0.9 0.7 5.4 65 0.1 26.9 0.23.8 19.2 1.5 35.7 9.1 1.7 0.8 0.6 5.5 12 0.2 31.7 0.2 3.6 15.9 1.7 30.512.8 1.6 0.9 0.7 5.5 28 0.1 31.4 0.2 3.5 13.5 1.7 32.7 13.7 1.5 0.8 0.65.6 26 0.1 31.4 0.2 3.5 13.5 1.7 32.7 13.7 1.5 0.8 0.6 5.8 19 0.1 30.50.2 3.7 14.9 1.6 31.7 13.7 1.7 0.9 0.7 5.9 30 0.1 30.4 0.2 3.7 21.3 2.231.2 7.4 1.6 0.8 0.7 5.9  6 0.1 37.5 0.2 4.4 10.5 1.7 31.9 10.6 1.5 0.70.6 6.0  4 0.1 34.2 0.2 3.9 11.9 1.7 32.6 12.5 1.4 0.6 0.5 6.1 42 0.130.6 0.2 4.5 17.3 1.8 32.7 9.2 1.7 0.9 0.7 6.3 45 0.1 31.6 0.2 3.9 18.21.8 30.4 10.5 1.6 0.8 0.6 6.6 56 0.1 26.8 0.2 4.2 20.0 1.5 34.3 8.7 1.91.0 0.8 6.7 43 0.1 28.5 0.2 3.8 18.6 1.6 34.1 9.6 1.7 0.9 0.6 7.1 32 0.128.1 0.2 3.4 16.8 1.8 35.5 10.6 1.6 0.8 0.6 7.2 70 0.1 26.3 0.2 3.5 25.51.8 31.0 8.9 1.3 0.6 0.5 7.4 69 0.1 30.9 0.2 4.0 15.7 1.7 31.7 12.9 1.50.7 0.5 7.4 61 0.1 31.0 0.2 4.0 16.4 1.6 34.1 9.5 1.5 0.7 0.5 7.5 20 0.133.3 0.1 3.8 11.7 1.6 31.4 14.8 1.5 0.8 0.6 7.8 53 0.1 33.1 0.1 3.8 18.21.9 29.8 10.4 1.3 0.6 0.5 8.4 18 0.1 29.4 0.2 3.7 18.4 1.7 32.8 10.9 1.40.6 0.5 9.1 51-1  0.1 29.0 2.0 3.6 17.1 1.6 33.8 9.9 1.4 0.7 0.5 9.2 470.1 30.5 0.1 4.2 20.3 1.5 31.9 8.3 1.5 0.7 0.5 9.3 51-60 0.1 30.7 2.63.4 15.8 1.9 31.2 11.6 1.3 0.7 0.5 10.2 46 0.1 24.8 0.1 3.6 28.8 1.630.3 7.9 1.3 0.6 0.5 10.2 48 0.1 33.1 0.1 3.8 16.5 1.7 30.4 11.4 1.4 0.70.5 10.7 51-13 0.1 25.4 2.2 3.3 23.8 1.6 32.7 8.3 1.3 0.6 0.4 11.3

TABLE 12 TAG levels (% leaf dry weight) and TAG composition in leaftissue from N. tabacum plants (T0 generation) expressing WRI1, DGAT1 andOleosin transgenes and supertransformed with a T-DNA encoding an SDP1hairpin construct (pOIL051). Leaf samples were harvested duringflowering. Line C14:0 C16:0 C16:1 C18:0 C18:1 C18:1d11 C18:2 C18:3n3C20:0 C22:0 C24:0 % TAG wild- 0.2 14.8 0.6 8.5 9.2 0.3 20.0 44.5 0.6 0.30.4 0.3 type 21 0.1 25.7 2.1 3.7 21.2 1.0 31.0 11.7 1.5 0.8 0.6 8.8 560.1 33.2 1.4 4.9 20.7 1.0 26.3 7.4 2.1 1.3 0.9 9.2 65 0.1 24.7 1.5 3.828.5 1.0 29.0 7.5 1.7 0.9 0.6 12.0 42 0.1 34.0 1.5 4.4 16.8 1.1 29.4 7.62.2 1.4 1.1 13.1 28 0.1 29.5 2.4 3.5 16.4 1.2 28.7 14.6 1.5 1.0 0.6 13.230 0.1 19.1 1.9 3.3 31.8 1.0 30.6 9.3 1.3 0.7 0.4 13.6 20 0.1 22.4 1.83.7 27.4 0.9 29.0 10.8 1.7 0.9 0.7 14.6 19 0.1 20.9 1.7 3.1 28.4 1.031.6 10.0 1.4 0.8 0.5 15.7 12 0.1 24.4 1.6 3.6 22.1 0.9 35.1 8.9 1.4 0.80.5 15.8 16 0.1 21.5 1.8 3.4 34.9 1.0 26.2 7.9 1.4 0.7 0.5 16.4 57 0.125.0 1.7 4.1 27.7 1.0 28.4 8.4 1.6 0.9 0.6 17.2 26 0.1 22.5 1.6 3.5 28.41.1 31.2 7.6 1.7 1.0 0.7 18.0 39 0.1 30.0 2.2 3.7 22.7 1.6 24.3 11.6 1.50.9 0.7 18.1 70 0.1 22.1 2.1 3.6 36.3 1.0 24.2 7.2 1.4 0.7 0.5 18.3 450.1 21.4 1.8 3.7 34.4 1.0 27.5 6.9 1.4 0.8 0.5 19.1 32 0.1 23.3 1.6 3.224.4 1.1 33.6 9.0 1.5 0.9 0.6 19.5 18 0.1 23.4 2.1 3.3 26.4 0.9 30.210.3 1.4 0.7 0.5 20.6   20Y 0.1 22.3 1.6 3.6 30.3 0.9 28.5 9.1 1.6 0.90.6 20.8 43 0.1 28.1 2.0 3.5 21.5 1.2 29.9 10.2 1.5 0.9 0.6 21.2  4 0.127.9 1.9 3.7 26.3 1.2 26.2 9.3 1.5 0.8 0.5 21.8  1 0.1 23.8 2.0 3.7 30.21.1 28.1 8.0 1.4 0.7 0.5 22.3 61 0.1 24.2 2.2 4.0 32.0 1.1 25.2 7.8 1.50.8 0.6 23.9 60 0.1 24.4 2.2 3.7 31.0 1.1 25.4 8.6 1.5 0.8 0.6 25.0 460.1 23.3 2.0 3.7 32.9 1.0 24.0 9.2 1.6 0.9 0.7 25.7  6 0.1 31.5 2.6 3.519.5 1.6 25.5 12.7 1.3 0.7 0.5 26.3 13 0.1 21.8 1.9 3.6 35.1 1.0 25.18.1 1.5 0.8 0.5 26.8 69 0.1 21.8 1.6 4.3 33.4 0.8 26.9 7.6 1.7 0.8 0.526.9 53 0.1 27.1 2.1 3.5 24.1 1.2 29.4 9.2 1.4 0.8 0.5 29.2 48 0.1 29.52.5 3.9 21.1 1.3 29.0 9.2 1.6 0.8 0.6 29.5 47 0.1 30.9 2.5 3.4 19.4 1.528.5 10.6 1.3 0.8 0.5 30.5

TABLE 13 TAG levels (% leaf dry weight) and TAG composition in leaftissue from N. tabacum plants (T0 generation) expressing WRI1, DGAT1 andOleosin transgenes and supertransformed with a T-DNA encoding an SDP1hairpin construct (pOIL051). Leaf samples were harvested at seed settingstage. Y = yellow leaf, G = green leaf. TAG content Sample C14:0 C16:0C16:1 16:3 C18:0 C18:1 C18:1d11 C18:2 C18:3n3 C20:0 C20:1d11 C22:0 C24:0(% dw) wt 0.0 13.0 0.0 0.0 8.4 7.0 0.0 24.7 46.8 0.0 0.0 0.0 0.0 0.3 730.0 26.6 1.7 0.0 8.5 9.4 0.0 27.0 25.6 1.1 0.0 0.0 0.0 0.6 18 0.1 15.01.8 0.0 4.8 14.4 0.4 43.9 16.3 1.7 0.4 0.7 0.5 3.3 41 0.1 22.3 1.2 0.34.4 24.0 0.6 32.8 10.8 1.7 0.3 0.8 0.5 5.2 19 0.1 14.5 1.5 0.3 3.0 21.60.7 44.8 10.6 1.4 0.4 0.7 0.4 7.2 20 0.1 26.7 2.4 0.1 4.3 24.9 1.0 25.211.3 1.9 0.3 1.0 0.8 9.6 30 0.1 18.6 1.5 0.3 3.5 24.8 0.7 38.9 8.9 1.40.3 0.7 0.4 9.9 65 0.1 22.2 1.4 0.3 3.5 30.9 0.7 29.1 8.1 1.7 0.3 1.00.6 11.3 42 0.1 23.6 1.5 0.2 4.1 29.0 0.9 30.3 5.9 2.0 0.4 1.3 0.8 12.032 0.1 21.3 1.3 0.3 3.3 21.4 0.9 40.7 7.1 1.7 0.3 1.0 0.6 13.7 39 0.125.8 1.7 0.3 3.6 27.2 1.2 27.2 8.2 2.0 0.4 1.4 0.9 14.0 45 0.1 23.0 1.50.1 3.8 28.0 0.9 32.6 6.3 1.8 0.3 1.0 0.6 14.4 13 0.1 26.9 2.8 0.1 3.732.6 1.1 21.6 7.6 1.6 0.3 0.8 0.7 14.6 R45   0.1 23.4 1.5 0.2 4.1 27.80.9 32.2 6.1 1.8 0.3 1.0 0.6 14.6 21 0.1 23.1 1.6 0.2 3.5 27.4 0.8 31.28.2 1.8 0.3 1.1 0.7 15.0  9 0.1 23.2 1.4 0.2 3.5 23.3 0.8 35.6 8.5 1.60.3 0.9 0.5 15.4 12 0.1 24.5 1.4 0.2 3.4 22.3 0.8 36.2 7.4 1.7 0.3 1.10.7 15.9  4 0.1 21.9 1.8 0.2 3.6 22.8 0.9 35.9 9.5 1.6 0.3 0.9 0.6 16.149 0.1 23.5 1.4 0.2 4.0 25.3 0.8 34.3 6.6 1.8 0.3 1.1 0.7 16.8 26 0.122.2 1.3 0.2 3.8 25.4 0.8 35.2 6.5 2.1 0.3 1.3 0.8 17.2 16 0.1 22.2 1.80.3 3.4 29.9 0.8 30.1 8.1 1.5 0.3 0.9 0.6 18.2  1 0.1 27.4 2.7 0.1 4.032.0 1.2 22.9 6.3 1.6 0.3 0.8 0.7 18.7 70 0.1 27.1 2.7 0.2 3.7 32.6 1.021.5 7.6 1.6 0.3 0.8 0.7 19.0  6 0.1 30.6 2.6 0.2 3.3 13.0 1.4 32.8 12.91.4 0.2 0.9 0.6 21.5 47 0.1 28.0 2.1 0.2 3.6 18.5 1.3 33.2 9.9 1.5 0.20.9 0.5 21.6 69 0.1 25.4 2.3 0.1 4.3 32.4 0.9 23.5 7.4 1.8 0.3 0.8 0.622.5 53 0.1 23.9 2.1 0.2 3.4 28.2 1.1 30.2 7.6 1.5 0.3 0.9 0.5 23.2 460.1 25.9 2.7 0.2 3.7 32.0 1.1 22.8 8.2 1.6 0.3 0.8 0.7 24.0 43 0.1 23.71.6 0.2 3.1 22.6 0.9 37.6 7.4 1.4 0.2 0.8 0.5 24.0 48 0.1 27.4 2.2 0.14.1 23.0 1.1 31.3 6.9 1.9 0.3 1.0 0.7 24.4 28 0.1 23.0 1.4 0.2 3.3 24.81.0 35.6 7.3 1.6 0.3 0.9 0.6 26.6   1Y 0.1 24.3 2.5 0.1 3.8 35.7 1.122.6 6.7 1.6 0.3 0.7 0.6 28.1   56G 0.1 25.5 1.8 0.2 3.7 26.7 0.9 29.87.3 1.8 0.3 1.1 0.7 33.9 57 0.1 25.1 1.9 0.2 3.2 20.1 1.0 35.2 10.0 1.50.3 0.9 0.6 35.4   56Y 0.2 24.8 1.4 0.2 4.1 27.2 0.8 31.0 6.0 2.0 0.41.2 0.8 39.6   69Y 0.1 24.7 2.1 0.2 4.1 32.0 0.8 24.3 7.8 1.9 0.3 0.90.7 46.5 R69Y 0.1 24.7 2.1 0.2 4.1 32.0 0.8 24.4 7.9 1.9 0.3 0.9 0.746.8 61 0.1 26.6 2.7 0.1 3.6 31.6 1.1 23.8 7.4 1.4 0.3 0.7 0.5 49.2  61Y 0.1 25.8 2.4 0.1 3.7 32.4 1.1 24.3 6.9 1.5 0.3 0.7 0.5 58.1 60 0.124.6 2.4 0.2 3.6 34.1 1.0 24.4 6.4 1.5 0.3 0.7 0.5 70.7

The selected T1 plants were grown in the glasshouse at the same time andunder the same conditions as control plants. Green leaf tissue samplesfrom the T1 plants before flowering were dried and total fatty acid(TFA) and TAG contents determined by GC analysis. TFA contents of theplants containing both T-DNAs ranged from 4.6% to 16.1% on a dry weightbasis including TAG levels in the same leaves of 1.2% to 11.8% on a dryweight basis (FIG. 9). This was much greater compared to the plantscontaining only the T-DNA from pJP3502 and growing alongside under thesame conditions and analysed at the same stage of growth, again showingthe synergism between reducing TAG lipase activity and the WRI1 plusDGAT combination. Plants containing only the pJP3502 T-DNA containedbetween 4.2% and 6.8% TFA including TAG levels of 1.4% to 4.1% on a dryweight basis (FIG. 9). Wild-type plants contained, on average, about0.8% TFA including less than 0.5% TAG on a dry weight basis. The fattyacid composition in the total fatty acid content and the TAG content ofleaves from each of lines #61 and #69 were similar to the composition inleaves containing only the T-DNA from pJP3502 (parent). Compared to thewild-type control leaves, plants containing both of the T-DNAs frompOIL51 and pJP3502 exhibited increased levels of C16:0, C18:1 and C18:2fatty acids. This significant shift in fatty acid composition camelargely at the expense of C18:3 which was reduced from about 50-55% toabout 20-30% as a percentage of the total fatty acid content.

The substantial increase in TFA levels including the TAG levels betweenthe plants containing only the pJP3502 T-DNA and plants containing theT-DNAs from both pOIL51 and pJP3502 was maintained throughout plantdevelopment. Control plants containing only the T-DNA from pJP3502contained 7.7% to 17.5% TAG during flowering while TAG levels rangedfrom 14.1% to 20.7% on a dry weight basis during seed setting. The TAGcontent in leaves from plants containing both pJP3502 and pOIL51 T-DNAsvaried between 6.3% and 23.3% during flowering and 12.6% and 33.6%during seed setting. Similar changes in fatty acid composition of theTAG fraction at both stages were detected as described earlier for thevegetative growth stage.

TAG levels were also found to be increased further in other vegetativetissues of the transgenic plants such as roots and stem. Some roottissues of the transgenic N. tabacum plants transformed with the T-DNAof pOIL051 contained 4.4% TAG, and some stem tissues 7.4% TAG, on a dryweight basis (FIG. 10). Wild-type plants and N. tabacum containing onlythe T-DNA from pJP3502 exhibited much lower TAG levels in both tissues.The addition of the hairpin SDP1 construct to decrease expression of theendogenous TAG lipase was clearly synergistic with the genes encodingthe transcription factor and biosynthesis of TAG (WRI1 and DGAT) forincreasing TAG content in the stems and roots. Of note, TAG levels inthe roots were lower compared to stem tissue within the same plant whilean inverse trend was observed in wild-type plants and N. tabacumcontaining only the T-DNA from pJP3502. The TAG composition of root andstem tissues exhibited similar changes in C18:1 and C18:3 fatty acids asobserved previously in transgenic leaf tissue. C18:2 levels in TAG werereduced in transgenic stem tissue while C16 fatty acids were typicallyreduced in transgenic root tissues when compared to the wild-typecontrol.

Therefore, the inventors concluded that addition of an exogenous genefor silencing the endogenous SDP1 gene to the combination of WRI1 andDGAT increased the total fatty acid content, including the TAG content,at all stages of the plant growth, and acted synergistically with WRI1and DGAT, particularly in the stems and roots. T1 seeds from thetransgenic plants were plated on tissue culture media in vitro at roomtemperature to test the extent and timing of germination. Germination ofT1 seed from three independently transformed lines was the same comparedto seed from the transgenic plants transformed with the T-DNA frompJP3502. Furthermore, early seedling vigour appeared to be unaffected.This was surprising given the role of SDP1 in germination in A. thalianaseeds and the observed defects in germination in SDP1 mutants (Eastmondet al., 2006). To overcome any germination defects if such had occurred,a second construct is designed in which the SDP1 inhibitory RNA isexpressed from a promoter which is essentially not expressed, or at lowlevels, in seed, such as for example a promoter from a photosyntheticgene such as SSU. The inventors consider that it is beneficial to reducethe risk of deleterious effects on seed germination or early seedlingvigour to avoid a constitutive promoter, or at least to avoid a promoterexpressed in seeds, to drive expression of the SDP1 inhibitory RNA.

It was noted that the T0 plants with the highest TAG levels had beengrown under high light conditions in the controlled environment room(500 micro moles light intensity, 16 hr light/26° C.-8 hr dark/18° C.day cycle) and appeared smaller (about 70% in height relative to theplants transformed with the T-DNA from pJP3502) than the wild-typecontrol plants. The inventors concluded that the combination oftransgenes and/or genetic modifications for the “push”, “pull”,“protect” and “package” approaches was particularly favourable forachieving high levels of TAG in vegetative plant parts. In this example,WRI1 provided the “push”, DGAT provided the “pull”, silencing of SDP1provided the “protect” and Oleosin provided the “packaging” of TAG.

Example 12. Senescence-Specific Expression of a Transcription Factor

Ectopic expression of master regulators of embryo and seed developmentsuch as LEC2 have been reported to increase TAG levels in non-seedtissues (Santos-Mendoza et al., 2005; Slocombe et al., 2009; Andrianovet al., 2010). However, constitutive over-expression of LEC2 in plantstransformed with a 35S-LEC2 gene resulted in unwanted pleiotropiceffects on plant development and morphology including somaticembryogenesis and abnormal leaf structures (Stone et al., 2001;Santos-Mendoza et al., 2005). To test whether limiting LEC2 expressionto the leaf senescence stage of plant development, i.e. after plants hadfully grown and reached their full biomass, would minimize undesirablephenotypic effects but still increase leaf lipid levels, a chimeric DNAwas designed and made for expression of LEC2 under the control of a A.thaliana senescence specific promoter from the SAG12 gene (U37336; Ganand Amasino, 1995).

To make the genetic construct, a 3.635 kb synthetic DNA fragment wasmade comprising, in order, an A. thaliana SAG12 senescence-specificpromoter, the LEC2 protein coding sequence and a Glycine max Lectin geneterminator/polyadenylation region. This fragment was inserted betweenthe SacI and NotI restriction sites of pJP3303. This construct wasdesignated pOIL049 and tested in leaves of N. tabacum plants which werestably transformed with genes encoding WRI1, DGAT1 and Oleosinpolypeptides, containing the T-DNA from pJP3502. UsingAgrobacterium-mediated transformation methods, the pOIL049 construct wasused to transform N. tabacum plant cells which were homozygous for theT-DNA of pJP3502 (Example 3). Transgenic plants comprising the genesfrom pOIL049 were selected by hygromycin resistance and were grown tomaturity in the glasshouse. Samples are taken from transgenic leaftissue at different stages of growth including at leaf senescence andcontain increased TAG levels compared to the N. tabacum pJP3502 parentline.

A total of 149 independent T0 plants (i.e. primary transformants) wereobtained. Upper green leaves of all plants and the lower brown, fullysenesced leaves of selected events were sampled at the seed settingstage of plant development and TAG contents were quantified by TLC-GC.The number of pOIL49 T-DNA insertions in selected plants was determinedby ddPCR using a hygromycin gene-specific primer pair. A TAG level of30.2% on a dry weight basis was observed in green leaf tissue harvestedat seed setting stage. TAG levels in brown leaves were lower in most ofthe plants sampled. However, three plants (#32b, #8b and #23c) displayedgreater TAG levels in brown senesced leaf tissue than in the greenexpanding leaves. These plants contained 1, 2 or 3 T-DNA insertions frompOIIL49.

T1 progeny of plants #23c and #32b were screened by ddPCR to identifynulls, heterozygous and homozygous plants for the T-DNA from pOIL049.Progeny plants of plant #23c containing zero T-DNA insertions frompOIL049 (nulls; total of 7) or two T-DNA insertions of the T-DNA frompOIL049 (homozygous; total of 4) were selected for further analysis.Similarly, progeny plants of plant #32b containing zero insertions(nulls; total of 6) or two insertions (homozygous; total of 9) weremaintained for further analysis. Green leaf tissue was sampled beforeflowering and TFA and TAG contents were determined by GC. Wild-typeplants and plants transformed with the T-DNA from pJP3502 were the sameas before (Example 11) and were grown alongside in the same glasshouse.TFA levels in leaves of the transformants containing the T-DNA frompOIL049 ranged from 5.2% to 19.5% on a dry weight basis before flowering(FIG. 12). TAG levels in the same tissues ranged from 0.8% to 15.4% on adry weight basis. This was considerably greater than in plantscontaining only the T-DNA from pJP3502. TAG levels in plants containingthe T-DNAs from pJP3502 and pOIL049 further increased to 38.5% and 34.9%during flowering and seed setting, respectively. When the fatty acidcomposition of the total fatty acid content was analysed for leaveshomozygous for the T-DNA from pOIL049, increased levels of C18:2 andreduced levels of C18:3 were observed (FIG. 12) while the percentages ofC16:0 and C18:1 remained about the same relative to leaves transformedonly with the T-DNA from pJP3502. These data demonstrated that theaddition of a second transcription factor gene under the control of anon-constitutive promoter to provide developmentally-regulatedexpression was able to further increase TAG levels in vegetative tissuesof a plant. The data also indicated that the senescence-specificpromoter SAG12 had some expression in the green tissue prior tosenescence of the leaves.

TAG levels were much increased in stem tissue when compared to bothwild-type N. tabacum plants and transgenic plants containing the T-DNAfrom pJP3502 alone. Some stem tissues of the transgenic N. tabacumplants transformed with the T-DNA from pOIL049 contained 4.9% TAG on adry weight basis (FIG. 11). On the other hand, TAG levels in root tissueexhibited large variation between the three pOIL049 plants with someroot tissues containing 3.4% TAG. Of note, TAG levels in roots werelower compared to stem tissue within the same plant while an inversetrend was observed in wild-type plants and N. tabacum containing onlythe T-DNA from pJP3502. The TAG composition of root and stem tissuesexhibited similar changes in C18:1 and C18:3 fatty acids as observedpreviously in transgenic leaf tissue. C18:2 levels in TAG were reducedin transgenic stem tissue while C16 fatty acids were typically reducedin transgenic root tissues when compared to the wild-type control.

Corresponding genetic constructs are made encoding other transcriptionfactors under the control of the SAG12 promoter, namely LEC1, LEC1like,FUS3, ABI3, ABI4 and ABI5 and others (Example 10). For example,additional constructs were made for the expression of the monocottranscription factor Zea mays LEC1 (Shen et al., 2010) or Sorghumbicolor LEC1 (Genbank Accession No. XM_002452582.1) under the control ofmonocot-derived homolog of the A. thaliana SAG12 promoter such as themaize SEE1 promoter (Robson et al., 2004). Further constructs are madefor expression of the transcription factors under developmentallycontrolled promoters, for example which are preferentially expressed atflowering (e.g. day length sensitive promoters), Phytochrome promoters,Chryptochrome promoters, or in plant stems during secondary growth suchas a promoter from a CesA gene. These constructs are used to transformplants, and plants which produce at least 8% TAG in vegetative parts areselected.

Starch and Sugar Levels

Starch and soluble sugar levels were measured in leaf tissue sampledfrom wild-type and transgenic plants containing the T-DNA from pJP3502,or the T-DNAs from both pJP3502 and pOIL51 or pJP3502 and pOIL049. Ingeneral, an inverse correlation was found between TAG and starch levelsin leaf tissue on a dry weight basis in the leaves having both T-DNAs(FIG. 13). In contrast, leaf soluble sugars levels were about the samein the transgenic plants relative to the wild-type, suggesting thatthere was no significant bottleneck in the conversion from sugars toTAG. An effect of the leaf position in the plant was observed inwild-type plants where starch levels tended to increase from lower leafto higher leaf position. No such effect was detected in the transgenicplants.

Example 13. Stem-Specific Expression of a Gene Encoding a TranscriptionFactor

Leaves of N. tabacum plants expressing transgenes encoding WRI1, DGATand Oleosin contain about 16% TAG at seed setting stage of development.However, the TAG levels were much lower in stems (1%) and roots (1.4%)of the plants (Vanhercke et al., 2014). The inventors considered whetherthe lower TAG levels in stems and roots were due to poor promoteractivity of the Rubisco SSU promoter used to express the gene encodingWRI1 in the transgenic plants. The DGAT transgene in the T-DNA ofpJP3502 was expressed by the CaMV35S promoter which is expressed morestrongly in stems and roots and therefore was unlikely to be thelimiting factor for TAG accumulation in stems and roots.

In an attempt to increase TAG biosynthesis in stem tissue, a constructwas designed in which the gene encoding WRI1 was placed under thecontrol of an A. thaliana SDP1 promoter. A 3.156 kb synthetic DNAfragment was synthesized comprising 1.5 kb of the A. thaliana SDP1promoter (SEQ ID NO: 175) (Kelly et al., 2013), followed by the codingregion for the A. thaliana WRI1 polypeptide and the G. max lectinterminator/polyadenylation region. This fragment was inserted betweenthe SacI and NotI sites of pJP3303. The resulting vector was designatedpOIL050, which was then used to transform cells from the N. tabacumplants homozygous for the T-DNA from pJP3502 by Agrobacterium-mediatedtransformation. Transgenic plants were selected for hygromycinresistance and a total of 86 independent transgenic plants were grown tomaturity in the glasshouse. Samples were taken from transgenic leaf andstem tissue at seed setting stage and contain increased TAG levelscompared to the N. tabacum parental plants transformed with pJP3502.

Example 14. Increasing Oil Content in Seeds

Several groups have reported increased TAG levels in seed tissue ofmaize, canola or Arabidopsis thaliana upon over-expression of individualgenes encoding WRI1 and DGAT1 (Shen et al., 2010; Liu et al., 2010;Weselake et al., 2008; Jako et al., 2001; reviewed in Liu et al., 2012).Recently, van Erp et al. (2014) explored the effect of WRI1 and DGAT1co-expression on seed oil content in A. thaliana. Absolute TAG levelsincreased from 38% in the wild type and empty vector control to 44% intransgenic lines. Silencing of the SDP1 TAG lipase in combination withWRI1 and DGAT1 over-expression further increased TAG levels up to 45%.Of note, while average seed weight was found to be increased, the numberof seeds per plant was lower compared to control plants.

A synthetic DNA fragment of about 14.3 kb in length and containing theopen reading frames coding for the M. musculus MGAT2, A. thaliana DGAT1,A. thaliana WRI1 and A. thaliana GPAT4 polypeptides under the control ofthe seed specific promoters from genes encoding FAEI, Conlinin1 andConlinin2 was synthesized and inserted as a NotI-PstI fragment intopJP3416. The resulting vector was designated pTV55 (FIG. 14). A seriesof derived vectors were constructed from pTV55 by sequential removal ofindividual expression cassettes, each step using restriction enzymedigestion followed by self ligation. The GPAT4 cassette was deleted byPacI digestion, resulting in pTV56. A subsequent digest with SrfIremoved the MGAT2 expression cassette, yielding pTV57. The WRI1 cassettewas deleted using the flanking SbfI restrictions sites, resulting inpTV58. Finally, the DGAT1 cassette in pTV58 was exchanged for the WRI1cassette by digestion with SrfI and PacI, followed by T4 DNA polymerasetreatment and ligation. The WRI1 cassette was excised from pTV57 usingSbfI and treated with T4 polymerase. Ligation of the blunt end WRI1cassette into the SrfI-PacI digested pTV58 backbone yielded pTV59. Eachvector contained the e35S (containing dual enhancer region)::PAT gene asselectable marker gene, providing resistance to BASTA.

In summary, the constructs contained the following combinations ofgenes:

pTV55: ProCnl1::MGAT2+ProCnl2::DGAT1+ProCnl1::GPAT4+

ProFAE1::WRI1;

pTV56: ProCnl1::MGAT2+ProCnl2::DGAT1+ProFAE1::WRI1;

pTV57: ProCnl2::DGAT1+ProFAE1::WRI1;

pTV58; ProCnl2::DGAT1;

pTV59: ProFAE1::WRI1.

The constructs pTV55-pTV59 were introduced separately into A.tumefaciens strain AGL1 and used to transform C. sativa (cv. Celine) bya floral dip method adapted from Liu et al (2012). Briefly, the freshlyopened flower buds were dipped in A. tumefaciens solution for 15 sec,wrapped in plastic film and left overnight in the dark at 24° C. afterwhich the plastic was removed. A total of 3-4 floral dips were performedbased on the quality of the flowers available. Plants were grown tomaturity and T1 seed were harvested. Following germination of the T1seed in soil, established T1 seedlings (7-10 days) were sprayed with0.1% BASTA herbicide (250 g/L glufosinate ammonium; Bayer Crop SciencePty Ltd, VIC Australia) to select for plants expressing the PATselectable marker gene. Surviving seedlings were separated andtransferred to fresh soil pots and grown until maturity in theglasshouse at 22±1° C. (day) and 18±1° C. (night). T2 seeds wereharvested and the oil content (which is at least 95% TAG) of 3independent batches of 50 mg seed of each line was measured by NMR (MQC,Oxford Instruments). Calibration samples were prepared with driedKimwipes papers containing known amounts of C. sativa seed oil in 10 mmdiameter NMR test tubes, to generate a range of oil content based on theweights of paper and oil. The calibration samples were sealed withparafilm, and maintained at 40° C. heating block minimal 1 hour beforeusing to allow for the oil to distribute uniformly in the tissue. Thecalibration samples were measured three times with a 0.55 Tesla magnetand 10 mm diameter probe operating at a proton resonance frequency of23.4 MHz for 16 seconds. Magnet temperature was maintained at 40° C.Seeds samples were first dried in a 105° C. oven overnight to ensure themoisture content was less than 5%. Samples were subsequently weighed,transferred to a 10 mm diameter glass tube and incubated at 40° C. for 1hr prior to NMR analysis. The oil content was measured in triplicate byNMR against the calibration, based on the mass weight.

Copy number of the T-DNA(s) inserted in each transformed line wasdetermined by digital PCR (dPCR). Genomic DNA was first digested withEcoRI and BamHI to ensure physical separation of T-DNAs in case ofmultiple insertions. The C. sativa LEAFY gene (lfy) was chosen as areference gene and the selectable marker as the target gene in a dPCRmultiplex reaction. Probes were labelled with either HEX (referencegene) or FAM (target gene). The amplification reaction conditions wereas follows: 95° C. for 10 min (ramping of 2.5° C./s), 39 cycles of 94°C. for 30 s (ramping 2.5° C./s) and 61° C. for 1 min (ramping 2.5°C./s), 98° C. for 10 min. After PCR amplification, fluorescence ofindividual droplets was measured in a QX100 droplet reader (BioRad) andthe copy number was calculated using the QuantaSoft software (version1.3.2.0, BioRad).

Transformation of C. sativa yielded multiple transgenic eventsexhibiting increased TAG levels in segregating T2 seeds compared to theuntransformed wild type control (FIG. 15). Interestingly, the highestTAG levels were obtained with pTV57 which contains the genes coding forWRI1 and DGAT1. The additional insertion of MGAT2 (pTV56) andMGAT2+GPAT4 (pTV55) resulted in slightly lower TAG levels compared topTV55. Copy number determination revealed 1 or 2 T-DNA insertions forthe pTV57 lines displaying the second highest and highest seed oilcontent, respectively (Table 14).

Homozygous T2 plants transformed with the T-DNA from pTV057 were alsocrossed with C. sativa plants transformed with genes for expression ofthe fatty acid desaturases and elongases required for the synthesis andaccumulation of DHA in seed (WO2013/185184). F1 seeds showed increasedoil content as measured by NMR compared to the C. sativa seedstransformed with the DHA construct and without the T-DNA of pTV57. TheDHA content in the seeds is determined by measuring the levels of DHA inthe TAG fraction compared to the C. sativa DHA parent plant. The totalDHA content of the seeds (mg DHA/g of seeds) containing both T-DNAs isincreased relative to the total DHA content of the seeds containing onlythe DHA construct.

TABLE 14 Oil content (%) in T2 seed and copy number (by digital PCR) ofC. sativa pTV57 transgenic events. pTV57 line Seed oil (%) Copy numberCMD29-1 36.97 ? CMD29-2 36.69 ? CMD29-3 35.16 1.02 CMD29-4 28.47 6.6CMD29-5 40.60 2 CMD29-6 37.86 4.68 CMD29-7 39.17 2.94 CMD29-8 39.880.947 CMD29-9 36.70 1.04 CMD29-10 37.19 0.935 CMD29-11 31.20 14.3CMD29-20 33.08 6

Example 15. Effect of Oil Body Protein Expression on TAG Accumulationand Turnover

N. tabacum plants transformed with the T-DNA of pJP3502 and expressingtransgenes encoding A. thaliana WRI1, DGAT1 and S. indicum Oleosin hadincreased TAG levels in vegetative tissues. As shown in Example 11above, when the endogenous gene encoding SDP1 TAG lipase was silenced inthose plants, the leaf TAG levels further increased, which indicated tothe inventors that substantial TAG turnover was occurring in the plantsthat retained SDP1 activity. Therefore, the level of expression of thetransgenes in the plants was determined. While Northern hybridisationblotting confirmed strong WRI1 and DGAT1 expression and some oleosinmRNA expression, expression analysis by digital PCR and qRT-PCR detectedonly very low levels of oleosin transcripts. The expression analysisrevealed that the gene encoding the Oleosin was poorly expressedcompared to the WRI1 and DGAT1 transgenes. From these experiments, theinventors concluded that the oil bodies in the leaf tissue were notcompletely protected from TAG breakdown because of inadequate productionof Oleosin protein when encoded by the T-DNA in pJP3502. To improvestable accumulation of TAG throughout plant development, several pJP3502modifications were designed in which the Oleosin gene was substituted.These modified constructs were as follows.

-   -   1. pJP3502 contains a gene (SEQ ID NO:176 provides the sequence        of its complement) encoding the S. indicum oleosin which was        poorly expressed. That gene has an internal UBQ10 intron which        might be reducing the expression level. To test this, a 502 bp        synthetic DNA fragment containing the S. indicum oleosin gene        and lacking the internal UBQ10 intron was synthesized and        inserted into pJP3502 as a NotI fragment, to substitute the        oleosin gene containing the intron in pJP3502. The resultant        plasmid was designated pOIL040.    -   2. The Rubisco small subunit (SSU) promoter driving expression        of the oleosin gene in pJP3502 was replaced by the constitutive        enTCUP2 promoter. To this end, a 2321 bp fragment containing the        enTCUP2 promoter, Oleosin protein coding region, G. max lectin        terminator/polyadenylation region and the first 643 bp of the        downstream SSU promoter driving wri1 expression was synthesized        and subcloned into the AscI and SpeI sites of pJP3502 resulting        in pOIL038.    -   3. A similar strategy was followed for the expression of an        engineered version of the S. indicum oleosin gene containing 6        introduced cysteine residues (o3-3) under the control of the        enTCUP2 promoter (Winichayakul et al., 2013). A 2298 bp fragment        containing the enTCUP2 promoter, Oleosin o3-3 protein coding        region, G. max lectin terminator/polyadenylation region and the        first 643 bp of the downstream SSU promoter driving wri1        expression was synthesized and subcloned into the AscI and SpeI        sites of pJP3502 resulting in pOIL037.    -   4. The NotI sites flanking the S. indicum oleosin gene in        pJP3502 were used to exchange the protein coding region for one        encoding peanut Oleosin3 (Accession No. AAU21501.1) (Parthibane        et al., 2012a; Parthibane et al., 2012b). A 528 bp fragment        containing the oleosin3 gene, flanked by NotI sites, was        synthesized and subcloned into the respective site of pJP3502.        The resulting vector was designated pOIL041.    -   5. Similarly, a 1077 bp NotI flanked fragment containing the        gene coding for the A. thaliana steroleosin (Arab-1) (Accession        No. AAM10215.1) (Jolivet et al., 2014) was synthesized and        subcloned into the NotI site of pJP3502, resulting in pOIL043.    -   6. The Nannochloropsis oceanic lipid droplet surface protein        (LDSP) (Accession No. AFB75402.1) (Vieler et al., 2012) was        synthesized as a 504 bp NotI-flanked fragment and subcloned into        the NotI site of pJP3502, yielding pOIL044.    -   7. Finally, the A. thaliana caleosin (CLO3) (Accession No.        O22788.1) (Shimada et al., 2014) was synthesized as a 612 bp        NotI flanked fragment and subcloned into pJP3502, resulting in        pOIL042.

Each of these constructs was introduced into N. benthamiana leaf cellsas described in Example 1. Transient expression of both pJP3502 andpOIL040 in N. benthamiana leaf tissue resulted in elevated TAG levelsand similar changes in the TAG fatty acid profile but pOIL040 increasedthe TAG level more (1.3% compared to 0.9%). Each of the constructspOIL037, pOIL038, pOIL041, pOIL042 and pOIL043 were used to stablytransform N. tabacum plants (cultivar W38) by Agrobacterium-mediatedmethods. Transgenic plants were selected on the basis of kanamycinresistance and are grown to maturity in the glasshouse. Samples aretaken from transgenic leaf tissue at different stages during plantdevelopment and contain increased TAG levels compared to wild-type N.tabacum and N. tabacum plants transformed with pJP3502.

Cloning and Characterisation of LDAP Polypeptides from Sapium sebifera

Oleosins are not highly expressed in non-seed oil accumulating planttissues such as the mesocarp of olive, oil palm, and avocado (Murphy,2012). Instead, lipid droplet associated proteins (LDAP) have beenidentified in these tissues that may play a similar role to that ofoleosin in seed tissues (Horn et al., 2013). The inventors thereforeconsidered it possible that oleosin might not be the optimal packagingprotein to protect the accumulated oil from TAG lipase or othercytosolic enzyme activities in vegetative tissues of plants. LDAPpolypeptides were therefore identified and evaluated for enhancement ofTAG accumulation, as follows.

The fruit of Chinese tallow tree, Sapium sebifera, a member of thefamily Euphorbiaceae, was of particular interest to the inventors as itcontains an oil-rich tissue outside of the seed. A recent study (Divi etal, submitted for publication) indicated that this oleoginous tissue,called a tallow layer, might be derived from the mesocarp of its fruit.Therefore, the inventors queried the transcriptome of S. sebifera forLDAP sequences. A comparative analysis of expressed genes in the fruitcoat and seed tissues revealed a group of three previously unidentifiedLDAP genes which were highly expressed in the tallow layer.

Nucleotide sequences encoding the three LDAPs were obtained by RT-PCRusing RNAs derived from tallow tissue using three pairs of primers. Theprimer sequences were based on the DNA sequences flanking the entirecoding region of each of the three genes. The primer sequences were: forLDAP1, 5′-TTTTAACGATATCCGCTAAAGG-3′ (SEQ ID NO: 245) and5′-AATGAATGAACAAGAATTAAGTC-3′ (SEQ ID NO: 246) AT-3′; LDAP2,5′-CTITTCTCACACCGTATCTCCG-3′ (SEQ ID NO: 247) and 5′-AGCATGATATACTTGTCGAGAAAGC-3′ (SEQ ID NO: 248); LDAP3, 5′-GCGACAGTGTAGCGTTT-3′ (SEQID NO: 249) and 5′-ATACATAAAATGAAAACTATTGTGC-3′ (SEQ ID NO: 250).

Analysis of the S. sebifera transcriptome revealed multiple orthologsfor each of the LDAP genes, including eight LDAP1, six LDAP2, and sixLDAP3 genes, with less than 10% sequence divergence within each genefamily. The putative peptide sequences were aligned and a phylogenetictree was constructed using Genious software (FIG. 16), together withLDAPs homologs from other plant species, including two from avocado(Pam), one from oil palm, one from Parthenium argentatum (Par), two fromArabidopsis(Ath), five from Taraxacum brevicorniculatum (Tbr), threefrom Hevea brasiliensis (Hbr), as presented in FIG. 16. The phylogenetictree was revealed that the SsLDAP3 shared greater amino acid sequenceidentity to the LDAP1 and LDAP2 polypeptides from avocado and the LDAPfrom oil palm, while the SsLDAP1 and SsLDAP2 polypeptides were moredivergent.

Genetic Constructs for Over-Expression of LDAP

In order to test the function of the LDAPs from S. sebifera, expressionvectors were made to express each of these polypeptides under thecontrol of the 35S promoter in leaf cells. The full length SsLDAP cDNAsequences were inserted into the pDONR207 destination vector byrecombination reactions, replacing the CcdB and Cm(R) regions of thedestination vector with the SsLDAP cDNA fragments. Followingconfirmation by restriction digestion analysis and DNA sequencing, theconstructs were introduced into Agrobacterium tumefaciens strain AGL1and used for both transient expression in N. benthamiana leaf cells andstable transformation of N. tabacum.

The expression of each of the three SsLDAP genes under thetranscriptional control of the 35S promoter in N. benthamiana leaves incombination with the expression of 35S::AtDGAT1 and 35S::AtWRI1 yieldedsubstantially higher levels of TAG accumulation relative to the cellsinfiltrated with the 35S::AtDGAT1 and 35S::AtWRI1 genes without the LDAPconstruct. The TAG level was increased about 2-fold above the TAG levelin the control cells. A significant increase in the level of α-linolenicacid (ALA) and a reduced level of saturated fatty acids was observed inthe cells receiving the combination of genes. relative to the controlcells.

Co-Localisation of YFP-Fused LDAP Polypeptides with Lipid Droplets inLeaf Cells

In order to characterise SsLDAPs in vivo and observe their dynamicbehaviour, expression constructs were made for expression of fusionpolypeptides consisting of the LDAP polypeptides fused to yellowfluorescent protein (YFP). For each fusion polypeptide, the YFP wasfused in-frame to the C-terminus of the SsLDAP. The full open readingframe of each of the three LDAP genes without a stop codon, at its 3′end, was fused to the YFP sequence and the chimeric genes inserted intopDONR207. Following confirmation of the resultant constructs byrestriction digestion and DNA sequencing, the constructs were introducedinto A. tumefaciens strain AGL1 and used for both transient expressionin N. benthamiana leaf cells and stable transformation of N. tabacum.Three days following infiltration of the leaf cells with the LDAP-YFPconstructs, leaf discs from the infiltrated zones were stained with NileRed, which positively stained lipid droplets, and observed under aconfocal microscope to detect both the red stain (lipid droplets) andfluorescence from the YFP polypeptide. Co-localisation of LDAP-YFP withthe lipid droplets was observed, indicating that the LDAP associatedwith the lipid droplets in the leaf cells.

Example 16. Modification of Fatty Acids in Different Lipid Pools inLeaves Accumulating High Levels of TAG

The inventors have described the production of increased levels of TAGin N. tabacum leaves by co-expression of transgenes encoding A. thalianaWRI1, A. thaliana DGAT1 and S. indicum Oleosin (Vanhercke et al, 2014).To explore if fatty acid modifications in different lipid pools thatexist in leaves were possible with co-expression of the WRI1 and DGAT1gene combination, transient expression experiments were carried out tosee if fatty acids in the acyl-CoA and acyl-PC pools could occur. In oneexperiment, expression of a transgene encoding A. thaliana fatty acidelongase (AtFae1) which elongates C18:1-CoA to C20:1-CoA was combinedwith genes encoding WRI1 and DGAT to test modification in the acyl-CoApool. In a second experiment, a transgene encoding A. thaliana fattyacid desaturase 2 (AtFAD2) which desaturates C18:1-PC to C18:2-PC wascombined with the WRI1 and DGAT genes to test modification in the PCpool. These experiments were designed to test the availability of theacyl substrates in the ER of the plant cells.

The gene encoding AtFAE1 was expressed from a CaMV35S promoter in N.benthamiana leaves separately or in combination with WRI1 and DGAT1 asdescribed in Example 1, in triplicate. Leaf samples were harvested 5days after infiltration with the Agrobacterium cells comprising thedifferent gene combinations. Total lipid was extracted from the leafsamples and the TAG fraction was separated from each by TLC. The fattyacid composition of each TAG fraction was determined and quantified withGC using a known amount of C17:0-TAG as an internal standard. As shownin Table 15, the C20:1 proportion in TAG was significantly increasedwhen AtAFE1 was expressed. Co-expressing WRI1 and DGAT1 with AtFAE1 alsoincreased the level of C20:1 product compared to the control, while thetotal TAG amount was increased from 0.8 to 14.9 μg/mg leaf. The C20:1product in TAG accumulated as high as 0.96 μg per mg, compared to 0.04μg per mg without the WRI1 and DGAT1 combination

TABLE 15 Modified fatty acid levels after transgenic expression ofmodifying enzymes in N. benthamiana leaves. TAG C20:1 C18:1 C20:1 (μg/mg(μg/mg Sample (%) (%) dw) dw) Control 6.9 ± 1.5 0.4 ± 0.4 0.4 ± 0.3 0.01WRI1 + DGAT1 17.1 ± 1.0  0.4 ± 0.2 16.3 ± 2.1  0.06 AtFAE1 7.0 ± 3.0 4.7± 3.1 0.8 ± 0.8 0.04 WRI1 + DGAT1 + 15.3 ± 0.5  6.4 ± 0.2 14.9 ± 2.5 0.96 AtFAE1

Similarly, AtFAD2 was co-expressed from the CaMV35S promoter in N.benthamiana leaves separately or in combination with WRI1 and DGAT1. Thefatty acid composition of the TAG fraction was determined and quantifiedas above. The level of C18:2 fatty acid in TAG was significantlyincreased when AtFAD2 was co-expressed with WRI1 and DGAT1, from 10.7%to 37.9%, and the level of C18:1 decreased from 19.5% to 7.6%. Inaddition, substantial levels of TAG (13.4 μg/mg leaf) were observed whenWRI+DGAT1+AtFAD2 were co-expressed in the leaves. These results clearlydemonstrated that the fatty acid composition and amount could bemodified by addition of fatty acid modification enzymes in combinationwith at least WRI1 and DGAT, and therefore could be used for increasedaccumulation of modified fatty acid products generated in either or bothof the acyl-CoA and PC pools, and eventually stored in TAG in thevegetative plant parts.

Example 17. Silencing of TGD Genes in Plants

Li-Beisson et al. (2013) estimated that in Arabidopsis leaves (a 16:3plant), approximately 40% of the fatty acids synthesized in chloroplastsenter the prokaryotic pathway, whereas 60% were exported to enter theeukaryotic pathway. After they were desaturated in the ER, about half ofthese exported fatty acids are returned to the plastid to supportgalactolipid synthesis for thylakoid membranes. The transport (import)of the fatty acids as DAG or phospholipids into the plastid involvesTGD1, a permease-like protein of the inner chloroplast envelope. TheArabidopsis ABC lipid transporter comprising TGD1, 2, and 3 proteins wasidentified by Benning et al. (2008 and 2009) and more recently by Rostonet al. (2012). This protein complex is localized in the innerchloroplast envelope membrane and is proposed to mediate the transfer ofphosphatidate across this membrane. TGD2 polypeptide is aphosphatidic-binding protein, and TGD3 an ATPase. A novel Arabidopsisprotein, TGD4, was identified by a genetic approach (Xu et al., 2008)and inactivation of the TGD4 gene also blocked lipid transfer from theER to plastids. Recent biochemical data indicate that TGD4 isphosphatidate binding protein residing in the outer chloroplast envelopemembrane (Wang and Benning, 2012).

Xu et al. (2005) described leaky tgd1 alleles in A. thaliana resultingin reduced plant growth and high occurrence of embryo abortion. Leaftissue of A. thaliana tgd1 mutants contained increased TAG levels,likely as cytosol oil droplets. In addition, elevated TAG levels werealso found in roots of tgd1 mutants. No difference in seed oil contentwas detected. Similar TAG accumulation in leaf tissue has been reportedfor A. thaliana tgd2 (Awai et al., 2006), tgd3 (Lu et al., 2007) andtgd4 mutants (Xu et al., 2008). All tgd mutant alleles were eithersufficiently leaky or severely impairing in plant development.

TGD1 Silencing

A silencing construct directed against the TGD1 plastidial importer wasgenerated based on a full length mRNA transcript identified in the N.benthamiana transcriptome. A 685 bp fragment was amplified from N.benthamiana leaf cDNA while incorporating a PmlI site at the 5′ end. TheTGD1 fragment was first cloned into pENTR/D-TOPO (Invitrogen) andsubsequently inserted into the pHELLSGATE12 destination vector via LRcloning (Gateway). The resulting expression vector was designatedpOIL025 and is transiently expressed in N. benthamiana to assess theeffect of TGD1 gene silencing on leaf TAG levels. The TGD1 hairpinconstruct is placed under the control of the A. niger inducible alcApromotor by subcloning as a Pml1-EcoRV fragment into the NheI(klenow)-SfoI sites of pOIL020 (below). The resulting vector, designatedpOIL026, is super-transformed into a homozygous N. tabacum pJP3502 lineto further increase leaf oil levels.

Further constructs are made for expressing hairpin RNA for reducingexpression of the TGD-2, -3 and -4 genes. Transformed plants areproduced using these constructs and oil content determined in thetransformants. The transformed plants are crossed with the transformantsgenerated with pJP3502 or other combinations of genes as describedabove.

Example 18. Expression of Gene Combinations in Potato Tubers

Construction of pJP3506

A genetic construct containing three genes for expression in potatotubers was made and used for potato transformation. This construct wasdesignated as pJP3506 and was based on an existing vector pJP3502(WO2013/096993) with replacement of promoters to provide fortuber-specific expression. pJP3506 contained (i) an NPTII kanamycinresistance gene driven by 35S promoter with duplicated enhancer region(e35S) as the selectable marker gene and three gene expressioncassettes, which were (ii) 35S::AtDGAT1 encoding the Arabidopsisthaliana DGAT1, (iii) B33::AtWRI1 encoding the Arabidopsis thalianaWRI1, and (iv) B33::sesame oleosin, encoding the oleosin from Sesameindicum. The nucleotide sequences encoding these polypeptides were as inpJP3502. The patatin B33 promoter (B33) was a tuber specific promoterderived from Solanum tuberosum, which was provided by Dr Alisdair Femie,Max Planck Institute of Molecular Plant Physiology, Potsdam, Germany. Acircular plasmid map of pJP3506 is presented in FIG. 17.

The S. tuberosum Patatin B33 promoter sequence used in the pJP3506construct was a truncated version having 183 nucleotides deleted fromthe 5′ end and 261 nucleotides deleted from the 3′ end relative toGenBank Accession No. X14483. The nucleotide sequence of the patatin B33promoter as used in pJP3506 is given as SEQ ID NO: 211.

Transformation of Potato

Potato seedlings (Solanum tuberosum) of cultivar Atlantic which had beengrown asceptically in tissue culture were purchased from Toolangi Elite,Victorian Certified Seed Potato Authority (ViCSPA), Victoria, Australia.Stem internodes were excised into pieces of approximately 1 cm in lengthunder a suspension of Agrobacterium tumefaciens strain LBA4404containing pJP3506. The Agrobacterium cells had been grown to an OD of0.2 and diluted with an equal volume MS medium. Excess Agrobacteriumsuspension was removed by brief blotting the stem pieces on sterilefilter paper, which were then plated onto MS medium and maintained at24° C. for two days (co-cultivation). The internodes were thentransferred onto fresh MS medium supplemented with 200 μg/L NAA, 2 mg/LBAP and 250 mg/L Cefetaxime. Selection of transgenic calli was initiated10 days later when the internodes were transferred onto fresh MS mediumsupplemented with 2 mg/L BAP, 5 mg/L GA3, 50 mg/L kanamycin and 250 mg/LCefetaxime. Shoots regenerated from calli were excised and placed ontoplain MS medium for root induction prior to transplanting into a 15 cmdiameter pot containing potting mix and grown in the greenhouse untilplant maturity including tuber growth.

DNA Extraction and Molecular Identification of the Transgenic Plants byPCR

Disks of about 1 cm in diameter were obtained from potato leaves fromthe plants in the greenhouse. These were placed in a deep-wellmicrotiter plate and freeze dried for 48 hr. The freeze dried leafsamples were then ground into powder by adding a steel ball bearing toeach well and shaking the plate in a Reicht tissue lyser (Qiagen) at amaximum frequency of 28/sec for 2 min each side of the microtiter plate.375 μL of extraction buffer containing 0.1 M Tris-HCl pH8.0, 0.05 M EDTAand 1.25% SDS was added to each well containing the powdered leaftissue. Following 1 hr incubation at 65° C., 187 μL of 6M ammoniumacetate was added to each well and the mixtures stored at 4° C. for 30min prior to centrifugation of the plates for 30 min at 3000 rpm. 340 μLsupernatant from each well was transferred into a new deep wellmicrotiter plate containing 220 μL isopropanol and maintained for 5 minat room temperature prior to centrifugation at 3000 rpm for 30 min. Theprecipitated DNA pellets were washed with 70% ethanol, air dried andresuspended in 225 μL H₂O per sample.

Two μL from each leaf sample DNA preparation was added to a 20 μL PCRreaction mix using the HotStar PCR system (Qiagen). A pair ofoligonucleotide primers based on 5′ and 3′ sequences from theArabidopsis thaliana WRI1 gene, codon-optimized for tobacco, was used inthe PCR reactions. Their sequences were: Nt-Wri-P3:5′-CACTCGTGCTTTCCATCATC-3′ (SEQ ID NO: 212) and Nt-Wri-P1:5′-GAAGGCTGAGCAACAAGAGG-3′(SEQ ID NO: 213). A pair of oligonucleotideprimers based on the Arabidopsis thaliana DGAT1 gene, codon-optimizedfor tobacco, was also used in a separate PCR reaction on each DNAsample. Their sequences were: Nt-DGAT-P2: 5′-GGCGATTTTGGATTCTGC-3′ (SEQID NO: 214) and Nt-DGAT-P3: 5′-CCCAACCCTTCCGTATACAT-3′ (SEQ ID NO: 215).Amplification was carried out with an initial cycle at 95° C. for 15min, followed by 40 cycles of 95° C. for 30 sec, 57° C. for 30 sec and72° C. for 60 sec. The PCR products were electrophoresed on a 1% agarosegel to detect specific amplification products.

Lipid Analysis of Potato Tubers

Thin slices of tubers harvested from regenerated potato plants, forconfirmed transgenic plants and non-transformed controls, werefreeze-dried for 72 hr and analysed for lipid content and composition.Total lipids were extracted from the dried tuber tissues usingchloroform:methanol:0.1 M KCl (2:1:1 v/v/v) as follows. The freeze-driedtuber tissues were first homogenized in chloroform:methanol (2:1, v/v)in an eppendorf tube containing a metallic ball using a Reicht tissuelyser (Qiagen) for 3 min at a frequency of 29 per sec. After mixing eachhomogenate with a Vibramax 10 (Heidolph) at 2,000 rpm for 15 min, ⅓volume of 0.1 M KCl solution was added to each sample and mixed further.Following centrifugation at 10,000 g for 5 min, the lower phasecontaining lipids from each sample was collected and evaporatedcompletely using N₂ flow. Each lipid preparation was dissolved in 3 μLof CHCl₃ per milligram of tuber dry weight. Aliquots of the lipidpreparations were loaded on a thin layer chromatography (TLC) plate (20cm×20 cm, Silica gel 60, Merck) and developed in hexane:diethylether:acetic acid (70:30:1, v/v/v). The TLC plate was sprayed withPrimuline and visualized under UV to show lipid spots. TAG and PL wererecovered by scraping the silica of the appropriate bands and convertedto fatty acid methyl esters (FAME) by incubating the material in 1 Nmethanolic-HCl (Supelco, Bellefonte, Pa.) at 80° C. for 2 hr togetherwith known amount of Triheptadecanoin (Nu-Chek PREP, Inc. USA) asinternal standard for lipid quantification. FAME were analysed by GC-FID(7890A GC, Agilent Technologies, Palo Alto, Calif.) equipped with a 30 mBPX70 column (0.25 mm inner diameter, 0.25 mm film thickness, SGE,Austin, USA) as described previously (Petrie et al., 2012). Peaks wereintegrated with Agilent Technologies ChemStation software (Rev B.04.03).

Among the approximately 100 individual transgenic lines regenerated,analysis of lipids derived from young potato tubers of about 2 cm indiameter revealed increased levels in total lipids, TAG andphospholipids fractions in tubers from many of the transgenic plants,with a range observed between no increase to substantial increases. Thefirst analysis of the potato tuber lipids indicated that a typicalwild-type potato tuber at its early stage of development (about 2 cm indiameter) contained about the 0.03% TAG on dry weight basis.

The content of total lipids was increased to 0.5-4.7% by weight (dryweight) in tubers of 21 individual transgenic plants, representing 16independently transformed lines (Table 16). Tubers of line #69 showedthe highest TAG accumulation at an average 3.3% on dry weight basis.This was approximately a 100-fold increase relative to the wild-typetubers at the same developmental stage. Tubers of the same transgenicline also accumulated the highest observed levels of phospholipids at1.0% by weight in the young tubers on a dry weight basis (Table 18). Theenhanced lipid accumulation was also accompanied by an altered fattyacid composition in transgenic tubers. The transgenic tubersconsistently accumulated higher percentages of saturated andmonounsaturated fatty acids (MUFA) and lower level of polyunsaturatedfatty acids (PUFA) in both the total fatty acid content and in the TAGfraction of the total fatty acid content (Table 17), particularly areduced level of 18:3 (ALA) which was reduced from about 17% in thewild-type to less than 10% in the transgenic tubers. The level of oleicacid (18:1) in the total fatty acid content increased from about 1% inthe wild-type to more than 5% in many of the lines and more than 15% insome of the tubers. Although palmitic acid levels were increased, thestearic acid (18:0) levels decreased in the best transgenic lines(Tables 16 and 17).

The transgenic potato plants were maintained in the glasshouse to allowfor continued growth of the tubers. Larger tubers of line #69 containedgreater levels of TFA and TAG than the tubers of about 2 cm in diameter.

Further increased levels of TFA and TAG are obtained in potato tubers byaddition of a chimeric gene that encodes a silencing RNA fordown-regulating the expression of the endogenous SDP1 gene, incombination with the WRI1 and DGAT genes.

Further Gene Combinations for Transformation of Potato

Total RNA from fresh developing potato (Solanum tuberosum L. cv.Atlantic) tubers was extracted by the TRIzol method (Invitrogen).Selected regions of the cDNAs encoding potato AGPase small subunit andSDP1 were obtained through RT-PCR using the following primers: st-AGPs1:5′-ACAGACATGTCTAGACCCAGATG-3′ (SEQ ID NO: 251), st-AGPa1:5′-CACTCTCATCCCAAGTGAAGTTGC-3′ (SEQ ID NO: 252); st-SDP1-s1:5′-CTGAGATGGAAGTGAAGCACAGATG-3′ (SEQ ID NO: 253), and st-SDP1-a1:5′-CCATTGTAGTCCTTTCAGTC-3′ (SEQ ID NO: 254). The PCR products were thenpurified and ligated to pGEMT Easy.

TABLE 16 Total lipid yield (% weight of potato tuber dry weight) and itsfatty acid composition in representative young potato tubers transformedwith the T-DNA of pJP3506, prior to flowering of the plants. Tubers ofline 65 were equivalent to the wild-type (non-transgenic) tubers. SampleC14:0 C16:0 C16:1 C16:3 C18:0 C18:1 C18:1d11 C18:2 C18:3 C20:0 C20:1C22:0 C24:0 % TFA  4-2 0.2 16.1 0.2 0.0 5.8 0.5 11.7 55.5 5.5 2.1 0.20.6 1.5 1.4 19 0.2 18.1 0.2 0.0 5.8 0.4 12.9 52.2 5.9 2.0 0.2 0.7 1.51.5 27-2 0.2 18.9 0.3 0.0 6.5 0.5 5.5 55.0 8.0 2.0 0.2 0.8 2.1 0.7 27-40.2 19.0 0.3 0.0 6.5 5.4 0.5 57.0 7.9 1.6 0.0 0.5 1.1 0.6 27-5 0.2 17.80.6 0.0 6.4 2.2 0.4 57.6 11.7 1.5 0.0 0.4 1.2 0.7 27-6 0.2 18.7 0.4 0.06.9 6.3 0.5 55.9 8.2 1.6 0.0 0.4 0.9 0.8 55 0.2 17.8 0.6 0.0 6.4 7.9 0.555.7 8.6 1.4 0.0 0.3 0.7 1.0 65 0.2 19.4 0.4 0.0 5.7 1.2 0.5 53.6 17.20.9 0.0 0.0 1.0 0.5 69 0.3 19.8 0.1 0.0 3.2 16.5 0.9 53.2 3.7 1.1 0.30.4 0.6 4.7 78 0.2 19.5 0.5 0.0 5.3 4.9 0.5 54.7 11.7 1.2 0.0 0.4 1.00.9 83 0.2 16.7 0.4 0.0 6.5 7.3 0.5 56.2 8.5 1.7 0.6 0.5 0.9 1.3 95-10.3 21.0 0.2 0.1 3.1 15.2 0.8 52.8 4.2 1.1 0.2 0.3 0.7 3.0 95-2 0.4 21.30.3 0.1 4.1 7.1 1.0 56.1 7.3 1.2 0.2 0.3 0.7 2.7 95-3 0.4 21.4 0.3 0.04.3 8.5 0.9 54.5 7.4 1.3 0.0 0.3 0.7 1.5 100  0.4 19.0 0.5 0.0 5.4 7.60.8 55.5 7.3 1.4 0.5 0.5 0.9 1.0 104  0.2 18.0 0.2 0.0 6.1 0.5 6.8 56.17.6 2.3 0.1 0.6 1.5 0.9 106  0.2 19.7 0.2 0.1 4.6 0.9 10.7 54.1 5.7 1.70.1 0.6 1.3 1.3

TABLE 17 TAG yield (% weight of potato tuber dry weight) and its fattyacid composition in representative young potato tubers, transformed withthe T-DNA of pJP3506, prior to flowering of the plants. Sample C14:0C16:0 C16:1 C16:3 C18:0 C18:1 C18:1d11 C18:2 C18:3 C20:0 C20:1 C22:0C24:0 % TAG WT 0.4 13.4 0.0 0.0 4.6 5.5 0.5 59.9 15.7 0.0 0.0 0.0 0.00.03  4-2 0.3 15.4 0.2 0.0 7.0 0.6 16.4 52.5 3.1 2.6 0.2 0.6 1.1 0.5 190.2 16.3 0.1 0.0 7.2 18.0 0.5 50.9 3.6 1.9 0.2 0.4 0.6 0.8 27-2 0.0 19.00.0 0.0 11.2 9.8 0.0 52.8 4.4 2.8 0.0 0.0 0.0 0.2 27-4 0.4 17.4 0.0 0.010.2 9.4 0.0 55.4 4.7 2.6 0.0 0.0 0.0 0.2 27-5 0.0 17.9 0.0 0.0 12.5 4.40.0 54.9 7.1 3.2 0.0 0.0 0.0 0.1 27-6 0.0 17.1 0.0 0.0 9.9 10.6 0.0 55.04.9 2.5 0.0 0.0 0.0 0.2 55 0.3 17.6 0.5 0.0 8.5 12.5 0.6 52.5 5.2 1.90.0 0.0 0.6 0.5 65 0.0 18.1 0.0 0.0 12.0 0.0 0.0 55.6 14.4 0.0 0.0 0.00.0 0.0 69 0.3 20.1 0.6 0.0 3.8 20.3 1.0 49.4 2.2 1.3 0.2 0.3 0.5 3.3 780.0 19.1 0.0 0.0 8.2 9.4 0.0 52.5 8.4 2.4 0.0 0.0 0.0 0.2 83 0.3 16.40.2 0.0 8.7 11.1 0.6 53.4 5.4 2.6 0.0 0.5 0.7 0.5 95-1 0.3 21.7 0.4 0.13.6 18.5 1.0 50.1 2.8 0.9 0.2 0.2 0.3 2.2 95-2 0.6 23.4 0.4 0.0 5.1 10.11.2 51.9 5.3 1.4 0.0 0.0 0.5 0.9 95-3 0.3 17.2 0.3 0.0 7.7 0.6 11.6 49.78.9 2.5 0.0 0.0 1.1 0.1 100  0.0 18.8 0.5 0.0 8.0 12.0 0.8 54.0 3.9 2.00.0 0.0 0.0 0.4 104  0.3 17.7 0.0 0.0 8.4 0.6 11.0 52.1 4.7 3.2 0.0 0.71.3 0.3 106  0.4 20.1 0.3 0.0 5.4 15.5 1.1 51.8 3.6 1.4 0.0 0.0 0.4 0.7

TABLE 18 Phospholipids yield (% weight of potato tuber dry weight) andits fatty acid composition in representative young potato tubers,transformed with the T-DNA of pJP3506, prior to flowering. Sample C14:0C16:0 C16:1 C16:3 C18:0 C18:1 C18:1d11 C18:2 C18:3 C20:0 C20:1 C22:0C24:0 % PL  4-2 0.2 21.2 0.2 0.0 4.6 0.4 3.8 57.8 9.3 0.9 0.0 0.0 1.70.3 19 0.1 22.7 0.2 0.0 4.4 5.1 0.3 54.9 8.9 0.7 1.0 0.5 1.2 0.4 27-20.2 21.0 0.3 0.0 5.2 2.8 0.4 56.9 9.3 0.9 1.3 0.4 1.4 0.4 27-4 0.0 22.90.0 0.0 6.0 2.3 0.0 57.2 8.8 1.1 0.0 0.0 1.6 0.3 27-5 0.0 19.6 0.5 0.05.0 1.2 0.0 58.7 12.6 1.0 0.0 0.0 1.4 0.4 27-6 0.0 22.9 0.0 0.0 6.3 2.60.0 56.3 9.3 1.2 0.0 0.0 1.5 0.3 55 0.1 21.2 0.4 0.0 5.1 2.1 0.0 57.811.4 0.7 0.0 0.0 1.0 0.4 65 0.0 21.4 0.4 0.0 5.9 1.1 0.0 53.2 15.7 1.00.0 0.0 1.3 0.3 69 0.2 21.5 0.2 0.0 2.3 3.7 0.6 61.9 7.9 0.6 0.0 0.4 0.81.0 78 0.0 22.1 0.4 0.0 4.4 2.7 0.4 55.6 12.2 0.8 0.0 0.0 1.3 0.4 83 0.221.1 0.3 0.0 5.0 2.9 0.4 57.1 10.7 0.8 0.0 0.4 1.1 0.5 95-1 0.2 24.8 0.50.0 2.6 3.5 0.6 59.1 7.6 0.6 0.0 0.0 0.6 0.6 95-2 0.3 22.1 0.0 0.0 2.72.1 0.6 61.0 9.6 0.7 0.0 0.0 0.9 0.6 95-3 0.2 23.2 0.5 0.0 3.1 3.6 0.757.7 9.3 0.7 0.0 0.0 0.9 0.5 100  0.0 23.3 0.5 0.0 4.6 3.0 0.4 57.2 9.00.8 0.0 0.0 1.1 0.4 104  0.0 21.3 0.0 0.0 4.8 2.7 0.0 58.3 10.1 1.0 0.00.0 1.7 0.4 106  0.2 23.2 0.2 0.0 3.8 3.0 0.6 57.1 8.6 0.7 1.0 0.4 1.10.4

Following verification by DNA sequencing, the cloned PCR products wereeither directly used as the target gene sequence to make a hairpin RNAiconstruct or fused by overlapping PCR. Three PCR fragments (SDP1,AGPase, SDP+AGP) were subsequently cloned into the pKannibal vector thatcontained specific restriction sites to clone the desired fragment insense and antisense orientation. The restriction sites selected wereBamHI and HindIII for cloning the fragment in the sense orientation andKpnI and XhoI for inserting the fragment in the antisense orientation.Primers sets used for amplification of the three target gene fragmentswere altered by addition of restriction sites which direct the fragmentinto cloning sites of pKannibal. The expression cassettes containing thetarget DNA fragment between the 35S promoter and OCS terminator inpKannibal were released with NotI and cloned into a binary vectorpWBVec2 with hygromycin as the plant selectable marker. Such binaryvectors were introduced into A. tumefaciens AGL1 strain and used forpotato transformation as described above.

Example 19. Increasing Oil Content in Monocotyledonous Plants

Expression in Endosperm

The oil content in the endosperm of the monocotyledonous plant speciesTriticum aestivum (wheat) and therefore in the grain of the plants wasincreased by expressing a combination of genes encoding WRI1, DGAT andOleosin in the endosperm during grain development usingendosperm-specific promoters. The construct (designated pOIL-Endo2)contained the chimeric genes: (a) the promoter of the GluI gene ofBrachypodium distachyon::protein coding region of the Zea mays geneencoding the ZmWRI1 polypeptide (SEQ IDNO:35)::terminator/polyadenylation region from the Glycine max lectingene, (b) the promoter of the Bx17 glutenin gene of Triticumaestivum::protein coding region of the A. thaliana gene encoding theAtDGAT1 polypeptide (SEQ ID NO:1)::terminator/polyadenylation regionfrom the Agrobacterium tumefaciens Nos gene, (c) the promoter of theGluB4 gene of Oryza sativa::protein coding region of the Sesame indicumgene encoding the Oleosin polypeptide::terminator/polyadenylation regionfrom the Glycine max lectin gene and (d) a 35S promoter::hygromycinresistance coding region as a selectable marker gene. The construct wasused to transform immature embryos of T. aestivum (cv. Fielder) byAgrobacterium-mediated transformation. The inoculated immature embryoswere exposed to hygromycin to select transformed shoots and thentransferred to rooting medium to form roots, before transfer to soil.

Thirty transformed plants were obtained which set T1 seed and containedthe T-DNA from pOIL-Endo2. Mature seeds were harvested from all 30plants, and 6 seed of each family cut in half. The halves containing theembryo were stored for later germination; the other half containingmainly endosperm was extracted and tested for oil content. The T-DNAinserted into the wheat genome was still segregating in the T1 seedsfrom these plants, so the T1 seeds were a mixture of homozygoustransformed, heterozygous transformed and nulls for the T-DNA. Increasedoil content was observed in the endosperm of some of the grains, withsome grains showing greater than a 5-fold increase in TAG levels. Theendosperm halves of six wild-type grains (cv. Fielder) had a TAG contentof about 0.47% by weight (range 0.37% to 0.60%), compared to a TAGcontent of 2.5% in some grains. Some families had all six grains withTAG in excess of 1.7%; others were evidently segregating with both wildtype and elevated content of TAG. In endosperms with elevated TAGcontent the fatty acid composition was also altered, showing increasesin the percentages of oleic acid and palmitic acid, and a decrease inthe percentage of linoleic acid (Table 19). The T1 grain germinatedwithout difficulty at the same rate as the corresponding wild-type grainand plants representing both high oil and low oil individuals from 14 T0families were grown to maturity. These plants were fully male and femalefertile.

The grain is useful for preparing food products for human consumption oras animal feed, providing grain with an increased energy content perunit weight (energy density) and resulting in increased growth rates inthe animals such as, for example, poultry, pigs, cattle, sheep andhorses.

TABLE 19 Fatty acid composition (% of total fatty acids) of TAG contentand the total TAG content (% oil by weight of half endosperms) intransgenic wheat endosperm Sample C14:0 C16:0 C16:1 C16:3 C18:0 C18:1C18:1d11 Control 1 0.3 16.9 0.1 0.0 1.6 15.6 0.6 Control 2 0.3 16.0 0.10.1 1.6 15.1 0.6 F5.3  0.1 20.1 0.1 0.1 2.6 23.5 0.6 F16.3 0.1 19.1 0.10.1 2.8 24.2 0.6 Sample C18:2 C18:3n3 C20:0 C20:1 C22:0 C24:0 % oil bywt. Control 1 60.4 4.0 0.1 0.4 0.0 0.0 0.5 Control 2 61.3 4.3 0.1 0.30.0 0.0 0.49 F5.3  48.5 2.4 0.8 0.7 0.3 0.4 2.5 F16.3 48.1 2.9 0.7 0.50.3 0.4 1.8

The construct pOIL-Endo2 is also used to transform corn (Zea mays) andrice (Oryza sativa) to obtain transgenic plants which have increased TAGcontent in endosperm and therefore in grain.

Expression in Leaves and Stems

A series of binary expression vectors was designed forAgrobacterium-mediated transformation of sorghum (S. bicolor) and wheat(Triticum aestivum) to increase the oil content in vegetative tissues.The starting vectors for the constructions were pOIL093-095, pOIL134 andpOIL100-104 (see Example 4). Firstly, a DNA fragment encoding the Z.mays WRI1 polypeptide was amplified by PCR using pOIL104 as a templateand primers containing KpnI restriction sites. This fragment wassubcloned downstream of the constitutive Oryza sativa Actin1 promoter ofpOIL095, using the KpnI site. The resulting vector was designatedpOIL154. The DNA fragment encoding the Umbelopsis ramanniana DGAT2aunder the control of the Z. mays ubiquitin promoter (pZmUbi) wasisolated from pOIL134 as a NotI fragment and inserted into the NotI siteof pOIL154, resulting in pOIL155. An expression cassette consisting ofthe PAT coding region under the control of the pZmUbi promoter andflanked at the 3′ end by the A. tumefaciens NOSterminator/polyadenylation region was constructed by amplifying the PATcoding region using pJP3416 as a template. Primers were designed toincorporate BamHI and SacI restriction sites at the 5′ and 3′ ends,respectively. After BamHI+SacI double digestion, the PAT fragment wascloned into the respective sites of pZLUbi1casNK. The resultingintermediate was designated pOIL141. Next, the PAT selectable markercassette was introduced into the pOIL155 backbone. To this end, pOIL141was first cut with NotI, blunted with Klenow fragment of DNA polymeraseI and subsequently digested with AscI. This 2622 bp fragment was thensubcloned into the ZraI-AscI sites of pOIL155, resulting in pOIL156.Finally, the Actin1 promoter driving WRI1 expression in pOIL156 wasexchanged for the Z. mays Rubisco small subunit promoter (pZmSSU)resulting in pOIL157. This vector was obtained by PCR amplification ofthe Z. mays SSU promoter using pOIL104 as a template and flankingprimers containing AsiSI and PmlI restriction sites. The resultingamplicon was then cut with SpeI+MluI and subcloned into the respectivesites of pOIL156.

These vectors therefore contained the following expression cassettes:

pOIL156: promoter O. sativa Actin1::Z. mays WRI1, promoter Z. maysUbiquitin::U. rammaniana DGAT2a and promoter Z. mays Ubiquitin::PAT

pOIL57: promoter Z. mays SSU::Z. mays WRI1, promoter Z. maysUbiquitin::U. rammaniana DGAT2a and Z. mays Ubiquitin::PAT.

A second series of binary expression vectors containing the Z. mays SEE1senescence promoter (Robson et al., 2004, see Example 4), Z. mays LEC1transcription factor (Shen et al., 2010) and a S. bicolor SDP1 hpRNAifragment were constructed as follows. First, a matrix attachment region(MAR) was introduced into pORE04 by AatII+SnaBI digest of pDCOT andsubcloning into the AatII+EcoRV sites of pORE04. The resultingintermediate vector was designated pOIL158. Next, the PAT selectablemarker gene under the control of the Z. mays Ubiquitin promoter wassubcloned into pOIL158. To this end, pOIL141 was first digested withNotI, treated with Klenow fragment of DNA polymerase I and finallydigested with AscI. The resulting fragment was inserted into theAscI+ZraI sites of pOIL58, resulting in pOIL159. The original RK2 oriVorigin of replication in pOIL159 was exchanged for the RiA4 origin bySwaI+SpeI restriction digestion of pJP3416, followed by subcloning intothe SwaI+AvrII sites of pOIL159. The resulting vector was designatedpOIL160. A 10.019 kb ‘Monocot senescence part1’ fragment containing thefollowing expression cassettes is synthesized: O. sativa Actin1:A.thaliana DGAT1, codon optimized for Z. mays expression, Z. mays SEE1::Z.mays WRI1, Z. mays SEE1::Z. mays LEC1. This fragment is subcloned as aSpeI-EcoRV fragment into the SpeI-StuI sites of pOIL160, resulting inpOIL161. A second 7.967 kb ‘Monocot senescence part2’ fragment issynthesized and contains the following elements: MAR, Z. maysUbiquitin::hpRNAi fragment targeted against S. bicolor/T. aestivum SDP1,empty cassette under the control of the O. sativa Actin1 promoter. Thesequences of two S. bicolor SDP1 TAG lipases (Accession Nos.XM_002463620; SEQ ID NO. 242 and XM_002458486; SEQ ID NO:169) and one T.aestivum SDP1 sequence (Accession No. AK334547) (SEQ ID NO: 243) wereobtained by a BLAST search with the A. thaliana SDP1 sequence (AccessionNo. NM_120486). A synthetic hairpin construct (SEQ ID NO:244) wasdesigned including four fragments (67 bp, 90 bp, 50 bp, 59 bp) of the S.bicolor XM_002458486 sequence that showed highest degree of identitywith the T. aestivum SDP1 sequence. In addition, a 278 bp fragmentoriginating from the S. bicolor XM_002463620 SDP1 lipase was included toincrease silencing efficiency against both S. bicolor SDP1 sequences.The ‘Monocot senescence part2’ fragment is subcloned as a BsiWI-EcoRVfragment into the BsiWI-FspI sites of pOIL161. The resulting vector isdesignated pOIL162.

The genetic constructs pOIL156 pOIL157, pOIL162 and pOIL163 are used totransform S. bicolor and T. aestivum using Agrobacterium-mediatedtransformation. Transgenic plants are selected for hygromycin resistanceand contain elevated levels of TAG and TFA in vegetative tissuescompared to untransformed control plants. Such plants are useful forproviding feed for animals as hay or silage, as well as producing grain,or may be used to extract oil.

Example 20. Extraction of Oil and Production of Biodiesel

Extraction of Lipid from Leaves

Transgenic tobacco leaves which had been transformed with the T-DNA frompJP3502 were harvested from plants grown in a glasshouse during thesummer months. The leaves were dried and then ground to 1-3 mm sizedpieces prior to extraction. The ground material was subject to soxhlet(refluxing) extraction over 24 hours with selected solvents, asdescribed below. 5 g of dried tobacco leaf material and 250 ml ofsolvent was used in each extraction experiment.

Hexane Solvent Extraction

Hexane is commonly used as a solvent commercially for oil extractionfrom pressed oil seeds such as canola, extracting neutral (non-polar)lipids, and was therefore tried first. The extracted lipid mass was 1.47g from 5 g of leaf material, a lipid recovery of 29% by weight. 1H NMRanalysis of the hexane extracted lipid in DMSO was preformed. Theanalysis showed typical signals for long chain triglyceride fatty acids,with no aromatic products being present. The lipid was then subjected toGCMS for identification of major components. Direct GCMS analysis of thehexane extracted lipid proved to be difficult as the boiling point wastoo high and the material decomposed in the GCMS. In such situations, acommon analysis technique is to first make methyl esters of the fattyacids, which was done as follows: 18 mg lipid extract was dissolved in 1mL toluene, 3 mL of dry 3N methanolic HCL was added and stirredovernight at 60° C. 5 mL of 5% NaCl and 5 mL of hexane were added to thecooled vial and shaken. The organic layer was removed and the extractionwas repeated with another 5 mL of hexane. The combined organic fractionswere neutralized with 8 mL of 2% KHCO3, separated and dried with Na2SO4.The solvent was evaporated under a stream of N2 and then made up to aconcentration of 1 mg/mL in hexane for GCMS analysis. The main fattyacids present were 16:0 (palmitic, 38.9%) and 18:1 (oleic, 31.3%).

FA 16:0 16:1 18:0 18:1 18:2 20:0 22:0 % wt 38.9 4.6 6.4 31.3 2.5 1.5 0.6Acetone Solvent Extraction

Acetone was used as an extraction solvent because its solvent propertiesshould extract almost all lipid from the leaves, i.e. both non-polar andpolar lipids. The acetone extracted oil looked similar to the hexaneextracted lipid. The extracted lipid mass was 1.59 g from 5 g of tobaccoleaf, i.e. 31.8% by weight. 1H NMR analysis of the lipid in DMSO wasperformed. Signals typical of long chain triglyceride fatty acids wereobserved, with no signal for aromatic products.

Hot Water Solvent Extraction

Hot water was attempted as an extraction solvent to see if it wassuitable to obtain oil from the tobacco leaves. The water extractedmaterial was gel like in appearance and gelled when cooled. Theextracted mass was 1.9 g, or 38% by weight. This material was like athick gel and was likely to have included polar compounds from theleaves such as sugars and other carbohydrates. The 1H NMR analysis ofthe material in DMSO was preformed. The analysis showed typical signalsfor long chain triglyceride fatty acids, with no aromatic products beingextracted. The left over solid material was extracted with hexane,yielding 20% of lipid by weight, indicating that the water extractionhad not efficiently extracted non-polar lipids.

Ethanol Solvent Extraction

Ethanol was used as an extraction solvent to see if it was suitable toobtain oil from the tobacco leaves. The ethanol extracted lipid wassimilar in appearance to both the water- and hexane-extracted lipid,being yellow-red in colour, had a gel-like appearance and gelled whencooled. The extracted lipid mass was 1.88 g from 5 g tobacco, or 37.6%by weight. The ethanol solvent would also have extracted some of thepolar compounds in the tobacco leaves.

Ether Solvent Extraction

Diethyl ether was attempted as an extraction solvent since it wasthought that it might extract less impurities than other solvents. Theextraction yielded 1.4 g, or 28% by weight. The ether extracted lipidwas similar to the hexane extracted material in appearance, wasyellowish in colour, and it did appeared a little cleaner than thehexane extract. While the diethyl ether extraction appeared to havegiven the cleanest oil, the NMR analysis showed a mixture of moreorganic compounds.

Production of Biodiesel from Tobacco Plants

A batch of transgenic tobacco plants was grown over winter (not itsnormal growing season) to assess oil production in the leaves during thecolder season with less natural light. The leaves from mature plants hadapprox 10% oil on a dry weight basis; much lower than plants grownduring the summer season. Nevertheless, lipid was extracted andconverted to biodiesel as follows. The stages in the process were: (a)extraction of crude lipid, (b) purification of TAG from the lipid, and(c) conversion of the purified TAG to biodiesel.

Hexane (petroleum ether 40-60 C) was used as the extraction solvent forobtaining oil comprising mostly non-polar lipid. 500 g of tobacco leafmaterial was dried, weighed and then soaked in hexane overnight withstirring. The mixture was filtered and the hexane extract was then driedwith magnesium sulphate and treated with active carbon to decolorise theoil. The solution was filtered and the resulting liquid evaporated in arotary evaporator, resulting in about 42 grams of crude oil. This wasyellow/green in colour and had a viscous consistency when cooled. Someof this oil was used in an attempt to make biodiesel directly but thenumber of impurities and high amount of free fatty acids gave rise tothe production of a lot of soap which hindered the methylation reactionand product separation. Therefore, further purification of this oil toenrich the TAG fraction was necessary prior to the transesterficationreaction to make biodiesel.

One problem with the winter grown sample was the presence of relativelyhigh levels of free fatty acids (FFAs) in the extracted material,resulting in excessive soap being made which hindered separation of themethyl esters and the glycerol products. To purify the TAG in the oil,several solvent systems were investigated and a hexane/ethyl acetatemixture of 80:20 was chosen as suitable for column chromatography.Separation on a silica column using hexane:ethyl acetate (80:20) wasperformed. The more hydrophobic TAG was the first to elute from thecolumn as an orange/yellow oil. Next eluted was a dark green band,containing a mixture of hexane-soluble components in the tobacco leavesincluding chlorophyl mixed with some TAG and FFAs. The final productwashed off the column with pure ethyl acetate and was mainly FFAs.

The purified TAG that was enriched away from FFAs and phosphates andother impurities could now be made into biodiesel. This was done byreacting the TAG with methanol in the presence of a base catalyst toproduce the methyl ester (biodiesel) and glycerol as a by product. Analternative method, acid-catalyzed esterification, can be used to reactfatty acids with alcohol to produce biodiesel in the presence of FFAs,requiring less pure TAG. Other methods such as fixed-bed reactors,supercritical reactors and ultrasonic reactors forgo or decrease the useof chemical catalysts and can also be used for biodiesel production fromlipids. However, base-catalyzed methods are the most economical forconverting purified TAG, requiring only low temperatures and pressuresand producing over 98% conversion yields provided the starting oil islow in moisture and free fatty acids.

The purified TAG was treated with methoxide solution (NaOH and methanolmixed until fully dissolved) at an oil temperature of 60° C. The mixturewas maintained at 60° C. and stirred for 2 hours as thetransesterification reaction took place. The reaction mixture was cooledto room temperature and two phases separated which were an upperbiodiesel layer and a lower glycerol layer. The phases were thenseparated using a separating funnel and the biodiesel recovered.

Hydrothermal Processing of High Oil Vegetative Tissues

Another, more direct approach to converting vegetative plant parts intoindustrial products such as liquid fuels is via hydrothermal processing(HTP). This was employed to convert the transgenic tobacco leaf materialcontaining about 30% TFA by weight into a renewable bio-oil that couldbe added to a conventional petroleum refinery feedstock to producerenewable diesel (paraffinic diesel). Petroleum diesel is a mixture ofmany hydrocarbon compounds, mainly alkanes, and is defined as being thefraction from the refinery between 200-300° C., typically comprisingpredominantly C13-C22 hydrocarbons. In a typical conversion oftransgenic tobacco leaf via HTP, the solid transgenic tobacco vegetativeplant material was mixed with water to create a solids concentrationbetween 16-50%. This slurry was then subject to temperatures between270-400° C. and 70-350 bar pressure. The reaction times varied between1-60 minutes and experiments were conducted with and without NaOH andKOH as catalyst.

Once the HTP processing had finished and reaction cooled, it wasseparated into 3 different product streams, namely gas, solid and liquidbio-oil. The bio-oil yields were between 25-40% on a dry weight basisrelative to the feedstock amount. FIG. 18 shows that much greaterbio-oil yields were obtained for the transgenic tobacco leaf materialrelative to the corresponding wild-type tobacco leaf material.

Direct In Situ Conversion of Lipid in Vegetative Plant Parts toBio-Diesel

In another series of experiments the water component of the HTP reactionwas replaced with the solvent methanol. There are a number of reasonsfor trying to use methanol, one being trying to convert TAG oil in theleaf directly (in situ) in one step to produce the methyl esters of thefatty acids (FAME) in the plant lipid and produce biodiesel directly.Using the same reaction conditions and equipment as the previous HTPexperiments, the water was replaced with methanol, the reactiontemperature was 335° C. at a pressure of 240 bar with NaOH as catalyst.The transgenic tobacco vegetative plant parts produced 47% bio-oil byweight relative to the input weight, while the wild-type tobaccoproduced 35% bio-oil by weight. H¹ NMR of the two resultant bio-oilsshowed only a small amount of FAME, while the NMR of the trangenictobacco bio-oil showed a large amount of the biodiesel FAME.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

All publications discussed and/or referenced herein are incorporatedherein in their entirety.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

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The invention claimed is:
 1. A process for producing extracted lipid,the process comprising i) extracting lipid from plant leaf piecesexpressing an exogenous Wrinkled 1 (WRI1) polypeptide and an exogenousdiacylglycerol acyltransferase (DGAT), and having inhibitedtriacylglycerol (TAG) lipase activity or expressing a Leafy Cotyledon 2(LEC2) polypeptide or both inhibited TAG lipase activity and expressinga LEC2 polypeptide, the leaf pieces having a surface area of at least 1cm² and a total non-polar lipid content of between 30% and 75% by weighton a dry weight basis, and ii) recovering the extracted lipid, therebyproducing the extracted lipid.
 2. The process of claim 1, wherein theleaf pieces in total have a dry weight of at least 2 g.
 3. The processof claim 1, wherein the leaf pieces have a total triacylglycerol (TAG)content of between 30% and 75% (w/w dry weight).
 4. The process of claim1 in which the volume of the extracted lipid is at least 1 liter.
 5. Theprocess of claim 1, wherein the step of extracting lipid from the leafpieces comprise one or more of rolling, pressing, crushing or grindingthe leaf pieces.
 6. The process of claim 1, wherein the leaf pieces areharvested from one or more plants at a time between about the time offlowering of the plants to about the time senescence of the plants hasstarted.
 7. The process of claim 1, wherein the leaf pieces have one ormore or all of the following features: i) oleic acid comprises at least19% of the total fatty acid content in the non-polar lipid in the leafpieces, ii) palmitic acid comprises at least 20% of the total fatty acidcontent in the non-polar lipid in the leaf pieces, iii) linoleic acidcomprises at least 15% of the total fatty acid content in the non-polarlipid in the leaf pieces, and iv) a-linolenic acid comprises less than15% of the total fatty acid content in the non-polar lipid in the leafpieces.
 8. The process of claim 1 which comprises one or more ofdegumming, deodorising, decolourising, drying and fractionating theextracted lipid, removing wax esters from the extracted lipid andanalysing the fatty acid composition of the extracted lipid.
 9. A leafpiece expressing an exogenous WRI1 and an exogenous DGAT, and havinginhibited TAG lipase activity or expressing a LEC2 polypeptide or bothinhibited TAG lipase activity and expressing a LEC2 polypeptide, andhaving a surface area of at least 1 cm² and a total non-polar lipidcontent of between 30% and 75% (w/w dry weight).
 10. A process forproducing a feedstuff, the process comprising admixing the leaf piece ofclaim 9 with at least one other food ingredient.
 11. A feedstuffcomprising the leaf piece of claim 9 and at least one other foodingredient.
 12. A process for producing a synthetic diesel fuel, theprocess comprising converting lipid in the leaf piece of claim 9 tosynthetic diesel fuel, or extracting lipid from the leaf piece of claim9 and converting the extracted lipid to the synthetic diesel fuel by aprocess comprising fractionation.
 13. A process for producing a biofuel,the process comprising converting the lipid in the leaf piece of claim 9to a bio-oil by pyrolysis, a bioalcohol by fermentation, or a biogas bygasification or anaerobic digestion.
 14. A process for feeding ananimal, the process comprising providing to the animal the feedstuff ofclaim
 11. 15. The process of claim 1, wherein the leaf pieces havereduced TAG lipase activity and express a LEC2 polypeptide.
 16. The leafpiece of claim 9, wherein the leaf piece has reduced TAG lipase activityand expresses a LEC2 polypeptide.
 17. The process of claim 2, whereinthe leaf pieces in total have a dry weight of at least 1 kg.
 18. Aprocess for producing a feedstuff, the process comprising extractinglipid from leaf pieces by the method of claim 1 and admixing theextracted oil with at least one other food ingredient.
 19. The processof claim 12, comprising a fractionation step in which hydrocarboncompounds are selected which condense between 150° C. and 200° C. orbetween 200° C. and 300° C.
 20. The process of claim 1, comprising astep of obtaining a plant which has been selected, or whose progenitorplant has been selected, on the basis of having in leaf pieces a totalnon-polar lipid content of between 30% and 75% by weight on a dry weightbasis.
 21. A process for producing the leaf piece of claim 9, comprisinga step of obtaining a plant which has been selected, or whose progenitorplant has been selected, on the basis of having in leaf pieces a totalnon-polar lipid content of between 30% and 75% (w/w dry weight).